DE69730814T2 - The NucA protein from Haemophilus influenzae and its coding gene - Google Patents

Abstract

A protein from H. influenzae designated NucA is isolated and purified. The NucA protein has the amino acid sequence of amino acids 26-603 of SEQ ID NO.2 or a biologically equivalent amino acid sequence thereof. Amino acids 1-25 of SEQ ID NO.2 are the signal peptide, which is cleaved during processing of the mature protein. The NucA protein has a molecular weight of approximately 63,000 Daltons as measured on a 12% SDS-PAGE gel and possesses 5'-nucleotidase activity. The NucA protein is obtained by isolation and purification from the H. influenzae organism, by chemical synthesis or by recombinant expression by an isolated and purified nucA DNA sequence which encodes the NucA protein. Such a DNA sequence hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the NucA protein of H. influenzae having the amino acid sequence of amino acids 26-603 of SEQ ID NO.2 or a biologically equivalent amino acid sequence thereof. The NucA protein is used to prepare a vaccine composition which elicits a protective immune response in a mammalian host to protect the host against disease caused by H. influenzae.

Description

Field of invention

These This invention relates to a 63,000 dalton protein produced by Haemophilus influenzae, designated NucA, and the nucA gene, which for this Protein encoded.

Background of the invention

Non-typable Haemophilus influenzae (NTHi) is a strictly human, commensal Organism, which in the upper airways of up to 80% of healthy Adult is found (bibliography entry 1). He is one premier Cause of otitis media and respiratory infections in children, including pneumonia and Sinusitis (2). There NTHi strains are not encapsulated, are the existing vaccines for H. influenzae, which are based on the capsule structure type b (Hib), not effective. Thus, a vaccine is needed specifically for NTHi organisms and potential vaccine components have been surface-exposed Concentrated antigens, such as outer membrane proteins.

Summary of the invention

Corresponding It is an object of this invention to provide an additional protein from H. influenzae to isolate and purify and test if the protein is a viable one Vaccine candidate in matching model systems.

It Another object of this invention is to isolate the gene and to clone which for encodes such a protein and express the protein recombinantly.

These and further objects of the invention as discussed below the isolation and purification of a protein of H. influenzae, which as NucA, as well as peptides of NucA protein, which an epitope or epitopes thereof. The isolated and purified NucA protein from H. influenzae has the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 or a biologically equivalent amino acid sequence from that. amino acids 1-25 of SEQ ID NO: 2 is the signal peptide which is generated during processing of the mature protein is split off. The NucA protein has a molecular weight from approximate 63,000 daltons as measured on a 12% SDS-PAGE gel, and has 5'-nucleotidase activity.

In an embodiment The invention provides the NucA protein by isolation and purification obtained from the H. influenzae organism. In a preferred embodiment According to the invention, the NucA protein is recombinantly isolated and purified nucA DNA sequence expressed for the protein coded.

The Invention includes an isolated and purified DNA sequence containing a DNA sequence which is under high stringency Southern hybridization standard conditions hybridized with a DNA sequence, which for the NucA protein of H. influenzae encoded with the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 or a biologically equivalent amino acid sequence from that.

Examples for such biologically equivalent NucA sequences are those wherein the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 by one or more of the following amino acid residue changes modified, selected from the group consisting of K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H or V436I.

The Invention includes also such a DNA sequence which under high stringency Southern hybridization Standard conditions with a DNA sequence with the nucleotide sequence from nucleotides 304-2037 of SEQ ID NO: 1 hybridized.

Around To obtain expression of the NucA protein, the DNA sequence is first in inserted a suitable plasmid vector. A suitable host cell is then transformed or transfected with the plasmid. In a embodiment this invention is the host cell Escherichia coli strain InvαF '. The host cell is then cultured under conditions involving the expression of allow said NucA protein by the host cell.

In another embodiment of this invention, the isolated and purified NucA protein or a peptide of NucA protein comprising an epitope or epitopes thereof is used to produce a vaccine composition which elicits a protective immune response in a mammalian host. The vaccine composition may further comprise an adjuvant, diluent or carrier. Examples of such adjuvants include aluminum hydroxide, aluminum phosphate, MPL ^™ , Stimulon ^™ QS-21, and IL-12. The vaccine composition is administered in an immunogenic amount to a mammalian host sufficient to protect the host from H. influenzae disease.

Short description of characters

1 outlined the KSCN extract of NTHi strain P860295 and its eluates from a Bio-Gel ^TM HT hydroxyapatite column run on a 12% SDS-PAGE gel. Lane 1 - BioRad's low molecular weight standards (apparent molecular weights of 97, 66, 45, 31 and 22 kD); Lane 2 - pre-column 3 M KSCN extract; Lane 3 - column flow; Lane 4 - 0.3 M NaPO ₄ , pH 7.0, eluate; Lane 5 - 0.4 M NaPO ₄ , pH 7.0, eluate.

2 outlined a 12% SDS-PAGE gel of the further purified NucA protein, showing that it is approximately 63 kD. Lane 1 - BioRad's low molecular weight standards (same as in 1 ); Lane 2 - concentrated NucA before ethanol precipitation.

3 outlined proposed nucA-specific PCR products amplified by the NTHi λ Zap library. The scheme of the λ Zap library shows the insertion site of P860295 genomic DNA fragments into the pBluescript ^™ region of the λ Zap ^™ II phage and the relative orientation of the nucA specific degenerate primers P1 and P2 to potential genomic DNA fragments containing these sites , The phage-specific T3 and T7 primer sites are shown fixed to the λ Zap ^™ II DNA.

4 (top) outlines the two PCR clones that contained most of the mature nucA sequence. From these sequences, a 420 bp probe constructed in the Southerns on P860295 chromosomal DNA was constructed. 4 (below) outlines a limited genomic restriction map of the nucA gene.

5 outlined a scheme of foreseen nucA-specific inverse PCR reactions and products. P860295 genomic DNA was digested with either NsiI (top) or EcoRI (middle and bottom). The DNAs were diluted and ligated to form "self-ligated" circles and the DNA circles containing nucA DNA are shown.

Above - the nearly 3 kb NsiI circle contained sequence information above the P1 nucA Region. PCR amplification using primer pairs 4313ext & 4102ext generated an approximate 2 kb DNA fragment.

Middle - The approximately 4.3 kb EcoRI circle would Sequence information above the P1 nucA region. PCR amplification under Use of primer pairs 4121fwd & 4122ext should be approximately 4 Generate kb DNA fragment. No DNA fragment of this size was observed.

Below - The nearly 1.6 kb EcoRI circle would Contain sequence information below the nucA coding region. PCR amplification using primer pairs 4102ext & 4313ext should be approximately 0.7 Generate kb DNA fragment. Indeed was an approximate 1.3 kb fragment obtained.

6 outlines the pRSETC expression vector, including the DNA sequence surrounding the cloning sites below the gene 10 T7 promoter (P _T7 ). The restriction enzyme DNA recognition sites, NdeI, NheI, BamHI and HindIII are underlined. The protein start and reading frame is shown under the DNA sequence, as well as the enterokinase cleavage site (EC cleavage).

7 outlines a Western blot of cell lysates from the expression products of pPX644 probed with polyclonal antibodies from rabbits. Lane 1: High molecular weight pre-stained protein standards (200, 97.4, 68, 43, 29, 18.4 and 14.3 kD) (Gibco BRL); Lane 2: NucA purified native protein standard; Lanes 3 & 4: BL21 (DE3) pLysS / pPX644, isolate no. 1, not tinduced or induced; Lanes 5 & 6: BL21 (DE3) pLysS / pPX644, isolate no. 5, uninduced or induced; Lanes 7 & 8: BL21 (DE3) pLysS / pPX644, isolate no. 7, uninduced or induced; Lanes 9 & 10: BL21 (DE3) pLysS / pRSETC, uninduced or induced. The arrow shows the migration of NucA.

8th (top) outlines a Coomassie stained 12% gel and 8th (bottom) outlines a Western blot of cell lysates of nucA mature sequence fusion clones # 1-3 of pPX693 showing uninduced and induced lysates probed with polyclonal antibodies from rabbits. A second clone of pPX707 (ompT signal sequence) is also outlined. Lane 1 - BRL high molecular weight protein standards with apparent molecular weights as shown in the Figure; Lane 2 - nNucA; Lane 3 - pPX707, clone # 2, uninduced; Lane 4 - pPX707, clone # 2, induced; Lane 5 - pPX693, clone # 1, uninduced; Lane 6 - pPX693, clone # 1, induced; Lane 7 - pPX693, clone # 2, uninduced; Lane 8 - pPX693, clone # 2, induced; Lane 9 - pPX693, clone # 3 (nucA in misalignment); Lane 10 - pPX693, clone # 3, induced no induction of the approximately 68 kD band; Lane 11 - pPX693, clone # 3, but at OD ₆₀₀ of 1.7 instead of 1.0; Lane 12 - lysate from an induced vector alone (pRSETC) culture, negative control; Lane 13 - a purified preparation of rNucA from pPX644; Lane 14 - empty; Lane 15 - BRL low molecular weight standards.

9 outlines the features of the pTrcHisC vector, along with the nucA signal and partially mature sequences, to show the insertion sites into the vector in the construction of pPX691. The first three sequence lines in 9 represent a partially native nucA sequence beginning at the first ATG of the signal sequence (SEQ ID NO: 22) and continuing into the mature sequence for 78 bases. The next two sequence lines represent the coding sequence of the vector (SEQ ID NO: 23) comprising the NcoI restriction site (containing the ATG start site), polyHis region, enterokinase cleavage site and the BamHI site of the multiple cloning region in the vector. The plasmid contains lacI ^q to suppress the highly inducible trc promoter so that expression is turned off until induction (there is a low basal level of leakage).

10 (top) outlines a Coomassie stained gel and 10 (bottom) outlines a Western blot of cell lysates of pPX691, pPX692 and pPX707, which are constructs with either the native (pPX691 and 692) or ompT (pPX707) signal sequence in different vectors and host strains. Included are negative controls of pRSETC, which is the vector for pPX692. The additionally induced lane (# 9 from the left) under pPX692 is from a culture induced at OD ₆₀₀ = 1.5, rather than 1.0, and shows a decrease in NucA protein expression. The uninduced and induced pRSETC lysates in lanes 10 and 11 are from a clone without nucA inserted, while the induced pRSETC lysate in lane 14 is from a vector that has been converted to a similar host strain. Lanes 1 and 15 - apparent molecular weight standards are listed on the figure (200, 97, 68, 43 and 29 kD; lane 2 - native NucA protein (but not enough protein was present to visualize with Coomassie staining) Lanes 3 and 5 - uninduced cell lysates from clones # 14 and # 15, respectively, of pPX691; lanes 4 and 6 - induced cell lysates from the same pPX691 clones; Lane 7 - uninduced cell lysate from clone # 11 from pPX692; Lanes 8 and 9 - cell lysates of the same culture but induced at OD ₆₀₀ of 0.9 and 1.5, respectively, lane 12 - pPX707, clone # 1, uninduced; lane 13 - pPX707, clone # 1, induced.

11 outlined a Coomassie gel expression of PelB and OmpT NucA fusion proteins. Lane 0 - pre-stained high molecular weight protein standards of apparent molecular weights as shown in the Figure; Lanes 1, 3, 5 & 7 - samples of supernatant; Lanes 2, 4, 6 & 8 - cell pellet samples; Lanes 1-4 - of BL21 (DE3) pLysS / pPX707 (OmpT-NucA); Lanes 5-8 - of BL21 (DE3) pLysS / pPX708 (PelB-NucA). The arrow shows the migration of NucA.

12 outlined a Coomassie-stained 12% SDS-PAGE gel on cell lysates of constructs with different signal sequences. Also included is the mature clone pPX709 with optimized TIR and the NTHi P860295 lysate. A negative control is the induced vector (pTrcHisC) alone. Here the vector contains no NucA sequence. Lanes 1 and 10 - molecular weight standards as above; Lane 2 - purified prep of rNucA from pPX691; Lane 3 - uninduced lysate of pPX691; Lane 4 - induced lysate of pPX691; Lane 5 - induced lysate of pPX707; Lane 6 - induced lysate of pPX708; Lane 7 - induced lysate of pPX709; Lane 8 - lysate of P860295; Lane 9 - induced pTrcHisC.

13 outlines the results of a 5'-nucleotidase assay on a TLC plate. Nucleotidase activity was determined with whole cells and purified native or recombinant NucA protein. The reactions contained the following additions: Lane 1: 10 μl P860295 cells; Lane 2: 5 μl rNucA; Lane 3: 1 μl rNucA; Lane 4: 5 μl nNucA; Lane 5: 1 μl of nNucA; Lane 6: no added nucleotidase; Lane C: 1 μl of 21.5 mM adenosine plus 1 μl of the reaction of lane 6 (control for migration of adenosine, shown as A). Solvent front is shown.

14 outlines the inhibition of rNucA (pPX691) 5'-nucleotidase activity by the anti-nNu cA Mab Nt-63-34-25. The inhibition of enzymatic activity is outlined in the absence (filled squares) or presence (hollow circles) of protein A beads.

15 outlined the dot-blot hybridization of several NTHi and type b, Eagan strain, as well as other species, probed with a nucA 420 bp dig-labeled probe. Numbers 1-22 are shown in Example 8 below.

detailed Description of the invention

These The invention relates to an isolated and purified H. influenzae NucA protein. The NucA protein has the amino acid sequence of amino acids 1-603 of SEQ ID NO: 2. Amino Acids 1-25 include the signal peptide. After normal processing, the mature NucA protein the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2. The NucA protein has a molecular weight of approximately 63,000 Daltons (63kD) as measured on a 12% SDS-PAGE gel. The NucA protein also has 5'-nucleotidase activity (See Example 5) which are monoclonal by an anti-native NucA antibody is inhibited (Example 6).

The NucA protein is present in very small amounts on the cell surface of H. influenzae present. NucA is a membrane-associated protein. This protein becomes high among all the H. influenzae strains tested preserved. The invention further relates to peptides of NucA protein, which comprise an epitope or epitopes thereof. Such peptides include or multiple epitopes associated with one or more epitopes of the NucA protein immunological are cross-reactive. Such peptides are generated first and then in terms of Cross-reactivity tested.

The NucA protein is isolated by isolation from the H. influenzae organism, by recombinant DNA expression or by chemical synthesis receive. Chemical synthesis involves using standard chemistries for festival and liquid phase synthesis a, in particular for the production of peptides from NucA protein, which is an epitope or epitopes thereof.

One Procedure for the isolation of the NucA protein from the H. influenzae organism is described in detail in Example 1 below. In summary the organism is extracted with KSCN, the extract is carried through two hydroxyapatite columns happened and subjected to SDS-PAGE. The band at 63 kD on the gel is excised to obtain purified NucA protein. example 4 describes in detail two alternative cleaning methods. The second hydroxylapatite column can through a MonoQ column be replaced. Alternatively, the KSCN extraction step is performed replaced a NaCl extraction step.

cloning of the gene which for encoding the NucA protein was not easy. Example 2 below represents the several strategies that were tried unsuccessfully, as well the ultimately successful cloning of the nucA gene, which the DNA sequence contains which for encodes the NucA protein. Successful cloning was followed by expression of the NucA protein and sequencing the nucA gene. The complete nucA gene sequence is shown in SEQ ID NO: 1, nucleotides 229-2037. The signal peptide becomes by nucleotides 229-303 coded. The mature NucA protein is encoded by nucleotides 304-2037.

example 3 below describes in detail the expression of the NucA protein. The full length nucA gene was cloned into an E. coli expression vector, pTrcHisC, and referred to as pPX691. The nucA gene became mature in E. coli NucA protein expressed is processed with its signal sequence. A Culture was with IPTG for Expression of the NucA protein. Expression was confirmed by both Western blot analysis, as well as Coomassie gel analysis confirmed.

These Invention further relates to an isolated and purified DNA sequence, which comprises a DNA sequence encoding the NucA protein of H. influenzae and accompanying DNA sequences encoding peptides of NucA protein encoding an epitope or epitopes thereof include.

A Variety of host cell vector systems is used to control the NucA protein and to express peptides of this invention, in addition to those described in U.S. Pat Example 3 will be described in detail. The vector system is compatible with the host cell used. Suitable host cells shut down Bacteria transformed with plasmid DNA, cosmid DNA or bacteriophage DNA; Viruses, such as cowpox virus and adenovirus; Yeast like Pichia cells; Insect cells such as Sf9 or Sf21 cells; or mammalian cell lines such as ovary cells Chinese hamsters; as well as other conventional organisms.

A variety of conventional transcriptional and translational elements can be used for the host cell len vector system. The nucA DNA is inserted into an expression system and the promoter and other control elements are ligated into specific sites within the vector such that when the plasmid vector is inserted into a host cell, the nucA DNA can be expressed by the host cell.

heterologous Signal peptides are used to recombinant NucA protein on the cellular membrane to translocate. Example 3 describes in detail the separate Clone above the mature nucA gene of the OmpT and PelB signal sequences. Expression of the NucA protein was with each of these signal sequences successful.

The Plasmid is produced by transformation, transduction or transfection introduced into the host cell, depending on the host cell vector system used. The host cell will then cultured under conditions which express the NucA protein allow through the host cell.

In addition to the DNA sequence obtained from NTHi strain P860295 which encodes the NucA protein (SEQ ID NO: 1), the present invention further comprises DNA sequences, which due to the redundancy of the genetic code to the sequences biologically equivalent are which for encode the NucA protein, that is those other DNA sequences characterized by nucleotide sequences which differ from those which are presented herein but which are for a protein or encode peptides with the same amino acid sequences as those for which by the DNA sequence of SEQ ID NO. 1 is encoded.

Especially considering the invention includes those DNA sequences which are derived from the sequence of SEQ ID No. 1 are sufficiently duplicative so as to hybridize with it under high stringency Southern hybridization standard conditions such as those described in Sambrook et al. (3) as well as the biologically active Enzymes.

These The invention also includes DNA sequences encoding amino acid sequences which differ from those of the NucA protein but which to those biologically equivalent are which for the protein will be described (SEQ ID NO: 2). Of such amino acid sequences can be said to be biologically equivalent to those of the NucA protein are, if their sequences only by smaller deletions of or conventional Substitutions to the NucA sequence differ so that the tertiary configurations opposite to the sequences those of the NucA protein are essentially unchanged.

To the Example may be a codon for the amino acid alanine, a hydrophobic amino acid, be substituted by one codon, which is another, less hydrophobic Rest encodes, such as glycine or a more hydrophobic residue, such as valine, Leucine or isoleucine. Similar can change, which substitution of a negatively charged residue for a others, such as aspartic acid for glutamic acid, or a positively charged rest for another, like lysine for Arginine, as well as changes based on similarities of Residues in their hydropathic index, also expected that they are a biologically equivalent product produce. Of nucleotide changes, what change the N-terminal or C-terminal portions of the protein molecule, were also not expected to alter the potency of the protein. each the proposed modifications are well within the routine skills according to the state of the art, such as determination of retention of biological Effectiveness of coded products. Therefore, when the terms "nucA-DNA" or "NucA-protein" in either of the Description or claims used They are each understood to carry all such modifications and include variations that involve the production of a biologically equivalent Protein.

specifically, As shown in Example 9 below, the NucA protein of this Invention one or more of the following changes in the amino acid sequence of SEQ ID NO: 2 have: K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H or V436I. One or more of these amino acid changes are present in other NTHi genomes and type b, Eagan strain.

The NucA protein and peptides of NucA protein containing an epitope or Epitopes of which include are in the manufacture of vaccines useful for protection of warm-blooded Animals to otitis media and pneumonia caused by H. influenzae to lend.

These Vaccine compositions include an isolated and purified NucA protein from H. influenzae or a peptide from NucA protein, which comprises an epitope or epitopes thereof, wherein the vaccine composition a protective one Immune response in a mammalian host triggers.

Vaccines containing the NucA protein or peptides can be mixed with immunologically acceptable diluents or carriers in a conventional manner to prepare injectable solutions or suspensions. In addition, the vaccines may include aluminum hydroxide, aluminum phosphate (alum) or other excipients such as Stimulon ^™ QS-21 (Cambridge Biotech Corporation, Worcester, MA), MPL ^™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc. , Hamilton, Montana), and IL-12 (Genetics Institute, Cambridge, MA).

The Vaccines of this invention may also additional H. influenzae include proteins which according to the state of Technique well known. Examples of such proteins are those which are referred to as P6 and P4 (the latter is also called Protein "e" known).

The Vaccines of this invention are made by injection into conventional Administered as subcutaneous, intraperitoneal or intramuscular injection in warm blooded Animals, as well as by oral administration and intranasal administration, to an effective immune response to protect against otitis media, caused through NTHi. The dose to be administered is determined by means comprising the Experts are known. Protection can be provided by a single vaccine dose or he may be administering multiple booster doses require.

Usually will be effective in the absence of human clinical data Leave immunization in a recognized animal model to the Predict the effectiveness of a vaccine in humans. It is suggested Chinchilla has been an animal model for effective immunization against NTHi (4) provides.

however showed the later ones effective immunization trials with NTHi protein candidates that the Chinchilla is not an acceptable model (unpublished data, Lederle practice Biologicals, West Henrietta, N.Y.).

The approved animal model for Hib consists of passive immunization of rat cubs (5) with bactericidal tests (6) in vitro. Active immunization is with the rat boy not possible. The rat boy is for only 4-5 Days prone for Hib; after that it is not prone. Due to this short period, it is not possible for the rat boy to be active to immunize because of the challenge with the bacterium more than five days must take place after immunization and the animal at the time not responded to challenge. Therefore, passive immunization looks with the Rat cubs the only feasible Animal model for Haemophilus before when taken together with bactericidal test data becomes.

In In the present invention, the NucA protein is shown to It is due to its effectiveness in both studies for passive Immunization in rat cows, as well as bactericidal tests (such as described in detail in Example 7 below) a useful vaccine candidate is. In the study with rat cubs, type b Eagan became the challenge strain used. The results showed a reduction in bacteremia in rats, which immunizes passively with anti-recombinant NucA protein serum had been.

In addition to The previous types of vaccine administration may be a technique used alternately as a genetic immunization, Polynucleotide immunization or naked DNA technology known is. Immunization with DNA has been used to challenge animals before challenge with influenza A to protect (7). In this technique, the nucA DNA is transformed into forms such as plasmid DNA and naked DNA are and will be used by the mammalian host in a variety of ways administered, including parenteral, mucosal and by gene-gun inoculation, such as by Fynan et al. described (8). Promotional agents such as bupivicain can used in this technique.

The NucA DNA is also used to prepare probes for use as Diagnosis in the detection of H. influenzae in selected clinical samples or laboratory strains to create. Clinical samples being analyzed include clinical Isolates, Oticus isolates and cervical cultures. The probes will be used in the diagnosis of meningitis, otitis media and pneumonia, which are caused by Hib or NTHi.

One such probe is the 420bp probe extending over nucleotides 416-835 of SEQ ID NO: 1. As described in Example 8 below, this 420 bp probe was used in a dot-blot hybridization assay. This probe was positive for all Haemophilus strains tested, including all NTHi strains and Type b, Eagan strain. The probe was negative for other bacterial strains including E. coli, Neisseria gonorrhoeae, Helicobacter pylori, Moraxella catarrhalis and Salmonella typhimurium. As expected For example, the probe was positive for an E. coli strain containing a rnucA plasmid. Thus, nucA probes are specific for nucA sequences and are useful for the diagnosis of the presence of H. influenzae strains.

rehearse of the E. coli strain InvαF ', which is the recombinant Plasmid pPX691 have been described by the Applicants to the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., and assigned ATCC entry number 98104 assigned. The plasmid pPX691 contains a nucA insert which for the NucA protein with the amino acid sequence from residues 26-603 coded by SEQ ID NO: 2.

The Material deposited with the ATCC may also be in connection with conventional Genetic engineering can be used to identify biologically equivalent sequences of the NucA protein including, but not limited to those in which the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 by one or more of the following amino acid residue changes modified, selected from the group consisting of K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H or V436I.

In order to This invention can be better understood, the following Examples shown. The examples are for the purpose of illustration only and are not to be considered as being the scope of the invention limit.

Examples

The following H. influenzae strains were used: NTHi strains P860295 Oticus isolate from Childrens's Hospital, Pittsburgh, PA P810384 Oticus isolate from Childrens's Hospital, Pittsburgh, PA P810568 Clinical isolate from Childrens's Hospital, Pittsburgh, PA P861454 Cervical culture from Childrens's Hospital, Pittsburgh, PA P880859 Opticus isolate from Childrens's Hospital, Pittsburgh, PA N1955 Clinical isolate from the University of Texas Southwestern Medical Center, Dallas TN106 Clinical isolate from the University of Texas Southwestern Medical Center, Dallas SH1013 Ear isolate from Texas SH1014 Ear isolate from Ohio SH1015 Blood isolate from Texas DL208 Clinical isolate from the University of Texas Southwestern Medical Center, Dallas N830161E Clinical isolate from the University of Texas Southwestern Medical Center, Dallas H305 Clinical isolate from Pittsburgh, PA Type b Eagan American Type Culture Collection, e.g. ATCC 31441 or 53763 Whittier Non-encapsulated type b from Childrens's Hospital, Boston, MA.

It standard molecular biology techniques used in accordance with the methods described in Sambrook et al. (3) described protocols.

example 1

Identification of NucA protein

All NTHi strains and the type b Eagan and Whittier strains were in BHI (Brain Heart Infusion) Broth grow left, added by 10 μg / ml Hemin stock (0.1 g hemin, 0.1 g histidine, 4 ml triethanolamine per 100 ml solution) and 40 μg / ml NAD or by 2% Fildes Enrichment (Remel, Lenexa, Kansas) and 20 μg / ml NAD. The cultures were at 37 ° C with shaking incubated.

A cell culture from NTHi strain P860295 was pelleted, washed with PBS or saline and ml in 10 of 3 M KSCN, 0.1 M PO _4, pH 6.0, per liter of culture and rocked at room temperature (RT) for 30 minutes , Unique bands (other than the outer membrane proteins (OMPs)) were seen when the KSCN extract was run on a 12% SDS-PAGE gel. One of these proteins ran at about 63 kD (see 1 ) and was further purified as described below.

The KSCN extract was purified by centrifugation in the Sorvall centrifuge at 8000 rpm. centrifuged for 20 minutes, then at 10,000 rpm. for 15 minutes in a SS-34 rotor. The supernatant was passed through a 0.22 μm filter and dialyzed against two changes of two liters each of 0.05 M sodium phosphate, pH 7.0. The dialyzed extract was loaded onto a Bio-Gel ^™ HT hydroxyapatite (HA) column (0.2-0.3 ml of HA per ml of extract) and equilibrated with 0.05 M phosphate, pH 7.0. The column was washed with about 10 column volumes of 0.05 M phosphate, pH 7.0. The 63 kD protein was step-eluted with about 5-6 column volumes of 0.3 M phosphate, pH 7.0, followed by a similar volume of 0.4 M phosphate, pH 7.0. The fractions containing the protein were pooled and concentrated in an Amicon stirrer cell using the YM30 membrane and particulates were collected at 3000 rpm. pelleted for 10 minutes. The fluid supernatant was dialyzed against 0.05 M phosphate, pH 7.0, equilibrated over a second HA column (approximately half the volume of the first column) and with 0.05 M phosphate, pH 7.0, for further purification , After the sample was loaded, the column was washed first with 0.05M phosphate, pH 7.0, then 0.3M phosphate, pH 7.0 (about 2 column volumes). The protein was eluted with a 0.3 M-0.4 M phosphate, pH 7.0, gradient (about 3 column volumes). Fractions were collected and all those containing the substantially pure 63 kD band (identified on the basis of size by SDS-PAGE gel) were pooled and concentrated.

Finally, 100 μg of protein eluted from a third HA column was repeatedly ultrafiltered in Centricon 30 to replace the phosphate with sterile water, ethanol precipitated and subjected to SDS-PAGE (see 2 ). The concentration was determined by the Peterson-Lowry method and N-terminal amino acid sequence analysis was performed. The first 26 amino-terminal residues have the sequence shown in residues 26-51 of SEQ ID NO: 2. Purification to homogeneity was achieved by electrophoresing the protein on a gel and then cutting out the 63 kD band. The protein was electroeluted from the gel disk, precipitated with ethanol, quantified, checked on SDS-PAGE and used in Immunogenicity Study # 1. See example 7 below.

A Animal study (study # 1) was performed to assess the immunogenicity of NucA protein to determine. See Tables 3, 5 and 6 below for the animal study data. antibody titers to this protein were measured. Whole cells (WC) and bactericidal (BC) tests were performed on the sera. From the native protein with the name "NucA" has been that it is antigenic and cross-reactive among all strains tested WC enzyme-linked immunosorbent assays (ELISA). In addition had the sera tested bactericidal efficacy for two out of four heterologous strains. Therefore, the NucA protein became a potential vaccine candidate considered. To larger quantities of the NucA protein would have to the gene for the protein encodes, first identified and cloned to to be expressed recombinantly.

Example 2

Cloning of the nucA gene

Preparatory steps

In carrying out this work, the following reagents and strains were used: DNA-modifying enzymes from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN); Agarose LE from Boehringer Mannheim; Sea Plaque ^TM low-melting agarose from FMC (Rockland, ME); Adenosine 5'-monophosphate and adenosine from Sigma (St. Louis, MO); Si250F TLC plates from JT Baker Chemical Co. (Phillipsburg, NJ); TA Cloning Kit, INVαF 'and pRSETC and pTrcHisC expression vectors from Invitrogen (San Dieago, CA); BL21 (DE3) pLysS, pET12a and pET20b from Novagen (Madison, WI); the λ Zap ^™ II vector and XL1-Blue MRF 'host strain of Stratagene (LaJolla, CA).

The standard methods of molecular biology used were similar to those of which by Sambrook et al. (3) was reported.

All NTHi strains and the type b Eagan and Whittier strains were in BHI (Brain Heart Infusion) Broth grow left, added by 10 μg / ml Hemin stock (0.1 g Hemin-0.1 g histidine-4 ml triethanolamine per 100 ml solution) and 40 μg / ml NAD, or by 2% Fildes enrichment and 20 μg / ml NAD. The cultures were at 37 ° C with shaking incubated.

Construction of a P860295 library in a λ Zap ^TM II vector

Approximately 50 μg (100 μl of P860295 chromosomal DNA was digested with 20 units of Tsp509I restriction enzyme (New England Biolabs [NEB]) in 10x buffer 4 (NEB) at 65 ° C for 50 minutes Fragments (4-10 kb) were grown isolated on a 0.7% Sea Plaque (FMC) agarose gel operating at 40 V for about 14 hours The bands of the appropriate size were cut from the gel, melted at 70 ° C, with a volume of 1 x TE, pH 7.5, diluted and extracted with an equal volume of warm (RT-37 ° C) phenol The supernatant was extracted with chloroform to remove phenol, one-tenth volume 3 M sodium acetate was added and the DNA was added to two volumes The DNA was pelleted, washed in 70% ethanol, dried and resuspended in 10 μl of sterile water Different amounts of DNA were digested on Eco-RI and dephosphorylated λ Zap ^™ II vector arms (Stratagene) at 12 ° C overnight lig The λ Zap ^™ II vector has been designed to allow in vivo excision and recircularization of cloned inserts contained within the vector to form a phagemid containing the cloned insert. Ligations were packed using Stratagene's extracts and plated on X11 Blue MRF 'cells. The efficiency of insertion varied from 90% to 95% based on white versus blue plaques on X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) plates. The white plaques contained inserts of about 4-6 kb.

plaques from the library were either monoclonal and polyclonal antibodies or with DNA probes either by hybridization with digoxigenin-11-dUTP (dig-dUTP) probes (Boehringer Mannheim) or in PCR reactions (see below).

Immunoblot screening of the λ Zap ^TM II library

The λ plaques were plated, about 500 pfu per 100 mm TY (5 g NaCl, 2 g MgSO ₄ .7H ₂ O, 5 g yeast extract, 10 g tryptone, per liter broth adjusted to pH 7.5 with NaOH, plus 15 g Difco Agar) plus isopropyl-beta-D-thiogalactopyranoside (IPTG) plates and incubated at 37 ° C overnight. The plates were cooled at 4 ° C for at least two hours. The plaques were blotted with dry Schleicher & Schuell (Keene, NH) NC ^™ nitrocellulose membranes for 5-10 minutes. The filters were blocked in 15 ml of 5% BLOTTO (in 1 x PBS) in 100 mm Petri dishes. Tween ^™ -20 (0.1-0.3%) was sometimes added to reduce background at 0.1-0.3%. The filter was shaken at RT for 4-6 hours or at 37 ° C for one hour. The BLOTTO was removed and primary antibody in 5% BLOTTO was incubated with the filter at 4 ° C overnight. The primary antibodies used were monoclonal antibodies Nt63-34-25, Nt63-60-14, Nt63 / 2-1-45 and Nt63 / 2-13-42 (generated as described in Example 7), or a mixture of these four monoclonal antibodies , or polyclonal rabbit serum, which had been absorbed into XL1-Blue MRF ', Y1090R', Y1089R 'and Y1088 cell cultures and λ Zap ^™ II lysate. Washes were performed at RT with 15 ml of 5% BLOTTO for 5-10 minutes each and repeated three times. Then, the filter was incubated with secondary antibody (KPL anti-mouse IgG + M No. QJ08-1, phosphatase-conjugated) diluted in 1: 1000 in 5% BLOTTO ± 0.1-0.3% Tween ^™ -20 and shaken for one hour at RT. Again, the filter was washed 3x with 15 ml each of 1x PBS + 0.1-0.3% Tween ^™ -20 at RT for 5-10 minutes. An additional wash was done in 1x PBS. The filter was then equilibrated in developing buffer (0.1M Tris, pH 9.5, 0.1M NaCl, 5mM MgCl ₂ ) for five minutes at RT. The filter was then placed in petri dishes containing 10 ml of development buffer plus 45 μl of NBT (nitro blue tetrazolium) and 35 μl of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and shaken to ensure that Filter immersed in developer. It was then kept in the dark on a flat surface until the spots were sufficiently dark. The reaction was stopped by rinsing the filter several times in deionized water and drying on 3MM Whatman paper in the dark.

There appeared to be a problem with the high background reactivity to λ Zap ^™ II plaques. The secondary antibody was found to be the major cause of background binding. Goat anti-mouse IgG + M lot # QJ08-1 (Kirkegaard & Perry Labs, Gaithersburg, MD), alkaline phosphatase-conjugated, had the lowest background among those tested when used at 1: 1000 dilution. Immunoblot screening revealed no positive clones for NucA. Due to the non-specific binding of the background, further work in this direction did not seem promising. Therefore, another strategy was used to try to extract nucA clones and sequences.

Plaque hybridization on λ Zap ^™ II library

Two 24-mer degenerate oligonucleotide probes were designed from the N-terminal sequence of the NucA protein (see Example 4 below) and assembled using an H. influenzae codon usage table for this purpose. The probes include amino acids 1-8 and 14-21 of the N-terminus of the mature protein and were named P1, and P2, respectively:
P1 AAAGAAGCACCACAAGCACATAAA (SEQ ID NO: 3)
P2 ATTTTACATATTAATGATCATCAT (SEQ ID NO: 4)

P1 and P2 were each wobbled at four bases. For P1, the A can be at residue 9 T, the A at residue 12 may be a T that A can be at residue 18 be a T, and the T to remainder 21 may be a C. For P2 that can T at residue 3 may be a C, the T at residue 9 may be a C, the T at Rest 12 may be a C and the T at residue 21 may be a C. Of the Primer P1 corresponds to nucleotides 304-327 of SEQ ID NO: 1 except that G at nucleotide 309 is an A in this primer. The primer P2 corresponds to nucleotides 343-366 of SEQ ID NO: 1, except, the G at nucleotide 348 in this primer is an A. The oligos were marked 3'-end with dig-dUTP according to the recommendations of the supplier.

The P1 and P2 probes were probed with genomic DNA by Southern hybridizations EcoRI and BamHI digested P860295 chromosomal DNA and EcoRI digested XL1-BlueMRF 'chromosomal DNA checked. at P860295 DNA became only one band with the P1 probe at approximately 4 kb is seen in EcoRI digestion while two bands at the P2 probe at approx 4 kb and 1.8 kb were seen. BamHI digestion of P860295 DNA showed an approximate 30 kb band. XL1-BlueMRF 'DNA did not cross-react with the probes. Prehybridization and hybridization incubations with P1 as a probe were at 53 ° C carried out; for the P2 probe they were at 45 ° C carried out. washes for P1 Hybridization was at 48 ° C carried out; washes for P2 were at 40 ° C carried out, Following standard protocols.

Plaque uplift were according to the procedure performed in Hybond-N (Amersham) log book. Prehybridization and Hybridization was at 42 ° C carried out, while washes at 45 ° C for P1 and 38 ° C were carried out for P2. Ten plates of library lysate, about 1000 pfu per 100 mm plate, were prepared using P1 or P2 screened as probes. Six supposedly positive ones were taken up, five from filter # 9 (P2 probe) and one from filter # 5 (P1 probe). These were saved and further screened by PCR. See below in the PCR screening section.

Phage library DNA cleaning

About 5 ml of the P860295 λ Zap ^™ II library phage were purified by a glycerol step gradient (9). The phage DNA was resuspended with 20 μl of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at approximately 1 ng / μl.

DNA purification

DNAs were either directly or from agarose gels using the LiCl protocol (10) or the GeneClean kit (Bio 101 Inc.) such as cleaned by the manufacturer. Plasmid mini-prep DNAs were added from 1 ml over Night cultures using Wizard Mini-Plasmid DNA Kit (Promega) or alkaline lysis-phenol standard extraction method isolated. Plasmid DNA in large scale was from 100-500 ml over Overnight cultures using the Maxi Plasmid DNA Kit (QIAGEN Inc.).

Partial DNA sequence from nucA

The basic principle for performing the following experiment was twofold: (1) The NTHi λ Zap ^™ II library was constructed from a partial Tsp509I genome digest, therefore separate size PCR bands should be prepared by amplifying the NTHi λ Zap ^™ II library using Combinations of phage spe primers T3 (SEQ ID NO: 16) and T7 (SEQ ID NO: 8) and nucA specific primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) as in 3 to be generated; and (2) PCR can be made to be less stringent and therefore more tolerant of nucleotide mismatches upon annealing of the primers. Primer T3 has the following sequence (which does not correspond to nucA):
T3 ATTAACCCTCACTARAGGGA (SEQ ID NO: 16)

Two DNA amplification reactions were performed, one with Taq DNA polymerase (Boehringer Mannheim) and the other with PFU DNA polymerase (Stratagene). Amplification cycles were as follows: denaturation, 95 ° C for 50 seconds; Annealing, 58 ° C for 60 seconds; Extension, 72 ° C for 4 minutes, 30 cycles, and then 72 ° C for 10 minutes. An aliquot of the reaction was electrophoresed in an agarose gel. Approximately 2000 bp and 600 bp DNA fragments were observed using T3 + P1 primers and T7 + P1 primers, respectively. The primer T7 (SEQ ID NO: 8) has the following sequence (which does not correspond to nucA):
T7 CGACTCACTATAGGGAGACC (SEQ ID NO: 8)

Both the approximately 600 bp and 2000 bp DNAs were gel isolated. An attempt was made to reamplete these DNAs as well as to repeat the previous PCR reaction with NTHi λ Zap ^™ II library DNA. The extension time in the PCR reaction was reduced to 72 ° C for two minutes. Only the PCR reaction with NTHi λ Zap ^™ II library DNA generated a PCR product of approximately 600 bp.

The approximately 600 bp amplified DNA was gel isolated and cloned into pCR ^™ II vector using the TA cloning kit as recommended by the supplier (Invitrogen); the resulting plasmid was named pPX640. The DNA sequence of the PCR insert was determined on an ABI 370 DNA sequencer using fluorescent dideoxynucleotide triphosphates and corresponds to nucleotides 304 to about 840 of SEQ ID NO: 1 with two base differences in the wobble positions of the P1 region. Nucleotide 309 is an A and nucleotide 315 is a T. The amino acid sequence derived from the 5 'end of the inserted DNA was identical to the N-terminal protein sequence of the native mature NucA protein described in Example 1. This confirmed the correct cloning of the partial nucA gene.

Similar PCR bands as above were obtained when PCR reaction was performed on the P2 pairs and the annealing temperature was reduced to 48 ° C. An approximately 1.7 kb band was amplified with the T3 primer pair and an approximately 600 bp band was seen with the T7 primer pair. These amplified fragments were similarly cloned into pCR ^™ II vector. Six white colonies were picked from each of P2 / T3 and P2 / T7 ligation. All had inserts when tested by EcoRI digestion. Approximately 600 bases and approximately 1.7 kb of sequences were obtained from the P1 / T7 clone and the P2 / T3 clone, respectively. The overlapping sequences of the two clones were similar, with expected changes in the P2 region because the P2 primer is a degenerate oligonucleotide. The P1 / T7 clone contained the 5'-536 bases of nucA and the P2 / T3 clone started below P1 and continued for about 1600 bases. The complete upper and lower sequences were not included in these clones.

PCR screening

Supposed positives of immunoblots (from the immunoblot screening of the λ Zap ^™ II library, supra) and oligonucleotide hybridization blots (from plaque hybridization, supra) were made using nucA primers 4101ext and 4102ext, SEQ ID Nos. 5 and 6, respectively Subjected to PCR amplification. These primers were generated during sequencing of the nucA region of pPX640 and have the following sequences:
4101ext CCAAAGTGGATATTGGTG SEQ ID NO: 5
4102ext CATCATAGAACTTCACATC SEQ ID NO: 6.

Of the Primer 4101ext corresponds to nucleotides 416-433 of SEQ ID NO: 1 Primer 4102ext corresponds to the complement of nucleotides 817-835 of SEQ ID NO: 1 in the reverse direction.

None the supposedly positive of immunoblots or oligonucleotide hybridization yielded the anticipated 420 bp band when using these two nucA primers was amplified.

Generation and marking the nucA 420 bp probe

The same sequencing primers, 4101ext (SEQ ID NO: 5) and 4102ext (SEQ ID NO: 6), were used to generate a 420 bp probe (see 4 above). Initially, the probes were generated from pPX640. Later, all labeled probes were prepared from P860295 genomic DNA. Use of chromosomal P860295 DNA reduced the background for the screening of λ Zap ^™ II clones. The 420 bp probe was labeled with a mixture of unlabelled nucleotides and varying concentrations of the labeled dig-dUTP nucleotide in a PCR reaction with Taq DNA polymerase (Boehringer Mannheim) dig-dUTP. Chromosomal P860295 DNA was used as template, providing a reduced λ Zap ^™ II background. PCR amplification was performed for 30 cycles, each operated at 95 ° C for 30-45 seconds (denaturation step), 42-52 ° C for 40-50 seconds (annealing step) and 72 ° C for 55-60 seconds (extension step). A hot start was performed on the genomic DNA at 95-98 ° C for 2-5 minutes. These PCR probes were precipitated in LiCl-ethanol and resuspended in 0.1% SDS in half to the same volume as the original volume, heated at 37 ° C for 10 minutes for better resuspension, and stored at -20 ° C. A 1: 500-1: 1000 dilution of the 420 hp probe was used in the hybridization. An estimate of the concentration was made by blotting 1 μl of each dilution, 10-2 to 10-6 on a strip of Hybond-N membrane along with known amounts of labeled standard and adjusting intensities after development.

Plaque hybridization with 420 bp probe

Plaque hybridizations were also performed using a dig-dUTP labeled 420 bp probe of pPX640 plasmid DNA (see above) according to the manufacturer's recommendations (Hybond-N membrane from Amersham, dig-dUTP for probe labeling from Boehringer Mannheim) using the standard protocol ( 3). However, this probe produced high background when tested with vector alone (λ Zap ^™ II) plaques. Potentially positive plaques from the λ Zap ^™ II library were identified and analyzed, but did not give the correct sized bands in a Southern hybridization experiment.

More library filters and a λ Zap ^™ II control filter were probed with a dig-dUTP labeled 420 bp probe generated from P860295 chromosomal DNA (see above). Two supposedly positives were identified, along with several questionable positives. The plaques were picked and plated for secondary screening. Re-probing showed some faint plaques. Phagemids were induced and digestion was performed on the DNAs. Southern hybridization was performed on the digest and the two putative positives showed some homology to the putative nucA sequence (from pPX643, see below). When subjected to PCR amplification with 4101ext (SEQ ID NO: 5) and 4102ext (SEQ ID NO: 6) primers, the two positives showed no amplification of the expected 420 bp band. The clones appeared to have limited homology to nucA and showed a different restriction pattern when compared to the PCR clone pPX643.

3 'end of nucA DNA

In order to obtain the DNA sequence from both the 5 'and 3' ends of the nucA gene (which are not present in the initial pPX640 construct), reverse PCR methodology was used, as shown schematically in FIG 5 shown. Southern analysis of NTHi genomic DNA digested with EcoRI or EcoRV and hybridized with the nucA specific 420 bp DNA probe gave the following results: For EcoRI digested genomic DNA, two bands were prepared at approximately 4.5 gb and 1.6 kb detected; For EcoRV digested DNA, a band at approximately 7 kb was detected. Ten-fold dilutions of these EcoRI and EcoRV-digested P860295 genomic DNAs were then self-ligated using T4 DNA ligase (NEB). These DNAs were amplified using reverse PCR methodology with Taq DNA polymerase. Amplification cycles were as follows: 95 ° C for 50 seconds, 52 ° C for 90 seconds, 72 ° C for 4 minutes, 30 cycles, and then 72 ° C for 10 minutes. An aliquot of the reaction was electrophoresed into an agarose gel. For the EcoRI ligated DNA, primers 4121fwd (SEQ ID NO: 17) GCTTTCAGCTAATGTGATTCC and 4122ext (SEQ ID NO: 18) CATCACAGCTGCATCTGCAG were used for the upper nucA DNA amplification reaction and primers 4313ext (SEQ ID NO: 7) and 4102ext (SEQ ID NO: 17). 6) amplification reaction was used for the lower nucA DNA. Primer 4121fwd corresponds to nucleotides 669-689 of SEQ ID NO: 1. Primer 4122ext corresponds to the complement of nucleotides 551-570 of SEQ ID NO: 1 in the reverse direction. Primers 4122ext and 4313ext were used with the EcoRV ligated DNA for both upper and lower nucA DNA amplification. An approximate 1.3 kb fragment was observed for the nucA EcoRI upper region. A faint approximately 2 kb fragment was observed for the EcoRV digested DNA. No DNA PCR product was observed for the nucA-EcoRI top region. Further attempts to amplify the 5 'end of the nucA gene from either of the genomic DNAs or from the NTHi λ Zap ^™ II library of DNA failed to produce either a DNA product or a DNA sequence by others Means of obtained nucA sequence corresponded. The approximately 1.3 kb PCR bottom product was directly sequenced as well as cloned into pCR ^™ II vector, and the plasmid was named pPX800. From the DNA sequence data, the complete 3 'end sequence, TAA stop codon and, additionally, lower non-coding sequence of the nucA gene were obtained.

Cloning of genomically "mature" nucA

To confirm and obtain the complete sequence of the mature nucA gene, a PCR reaction using the P1 degenerate (SEQ ID NO: 3) and 63K Revers (Rev) (SEQ ID NO: 9) primer to non-digested P860295 genomic DNA performed. The 63K Rev primer has the following sequence, which is the complement of nucleotides 2095-2114 of SEQ ID NO: 1 in the reverse direction:
63K Rev GAAGTCTTCAAACCTAGGAC (SEQ ID NO: 9).

Approximately 1 μg of P860295 genomic DNA was used in a PCR reaction consisting of the Taq DNA polymerase and these two primers. An aliquot of the reaction was electrophoresed in an agarose gel. The DNA fragment of the expected size of approximately 1.8 kb was observed. This DNA fragment was gel purified and cloned into pCR ^™ II and the plasmid was named pPX643. The DNA sequence of nucA in pPX643 was determined and corresponded to the sequence of previous partial data sets. This clone contains the complete mature nucA sequence but lacks the upper promoter region and signal sequence.

5 'Upper sequence of nucA using from PCR

Several attempts have been made to amplify the promoter region of DNA prepared from the P860295 λ library using the T3 and T7 primers coupled to several different nucA primers synthesized during the sequencing of P1 / P7 and P2 / T3 clones , None of these reactions produced the required fragment. Attempts to amplify this region by inverse PCR from the EcoRI 4.3 kb fragment were also unsuccessful (see above). Additional restriction fragments, preferably smaller than the 4.3 kb fragment, were therefore considered for inverse PCR. Southerns were performed on P860295 genomic DNA using the dig-dUTP labeled 420 bp chromosomal probe and restriction enzymes PstI, SalI, EcoRV, BamHI, ApaI, AvaI, KpnI, SacI, SmaI, XbaI, XhoI and At-rich enzymes BglII, HindIII, NsiI, SnaBI, SspI and PacI. From these hybridizations a limited genome restriction map was obtained (see 4 ).

NsiI digestion yielded an approximately 3 kb band that hybridized to the nucA 420bp probe; this is one of the smaller pieces identified. Therefore, this 3 kb fragment was gel-isolated from the genome digest, self-ligated and prepared for reverse PCR. An approximately 2 kb fragment was obtained upon amplification of the self-ligated NsiI fragment with the 4102ext (SEQ ID NO: 6) and 4313ext (SEQ ID NO: 7) primers. The primer 4313ext (SEQ ID NO: 7) has the following sequence corresponding to nucleotides 1762-1779 of SEQ ID NO: 1:
4313ext GTGAGTGTTGAAGTCTTG (SEQ ID NO: 7).

The fragment was sequenced and cloned into the pCR ^™ II vector. Eleven out of twelve colonies had inserts; ten were in one orientation (and were named pPX679) and one was in the opposite orientation (and was named pPX680). DNA sequencing of the insert within pPX680 produced about 1020 bases of additional sequence above the mature nucA gene. Thus, the clone contains the promoter region and signal sequence of the nucA gene. The sequence in the overlapping region corresponded to the sequence of previous clones (pPX640, the P2 / T3 clone and pPX643, described above), confirming the clone as nucA. See also SEQ ID NO: 1.

Cosmid Library construction

A Cosmid library was also constructed as follows: Genomic DNA from P810384 and P860295 was partially digested with Sau3AI to generate 30-50 kb fragments which in the BamHI site ligated from SuperCosI vector, a plasmid constructed from λ DNA and contained two integration sites (Stratagene). The used Host strain was NM554 (Stratagene). Colonies were treated with an anti-NucA monoclonal antibody (Mab Nt63 / 2-13-42), polyclonal antibody and dig-labeled DNA Probes screened.

Immunoblot screening from Cosmid Library

Mab Nt63 / 2-13-42 did not respond well to the P810384 strain on colony blots. Therefore, the screening at the P810384 library was stopped. Several supposed positives were identified from the P860295 library. The supposed positives were struck out and screened again, but they were of medium intensity. One Western was run on these positives (overnight cultures) and probed with Mab Nt63-34-25, which strongly responds to denatured NucA. However, no NucA bands were seen on the Westerns. Therefore, as in the λ Zap ^TM II library, immunoblot screening of the cosmid library was discontinued.

Colony hybridization at the cosmid library

Became similar the P860295 cosmid library screened using the 420 bp dig-labeled probe. This Screen generated 26 supposedly positives from the five library filters were identified. They were screened again and only four were positive of which two were very positive. Four isolated colonies were picked from one of the strong positives and were DNA preps for Southern Analysis prepares. DNA from two colonies gave similar hybridization restriction patterns in EcoRI, HindIII and ScaI & EspI Digestion while the other two colonies yielded DNAs of different sizes, one with a larger total insert size and one smaller. When the filter is at 70 ° C was washed, the two similar colonies gave only background binding. The other two clones showed positive hybridization at 70 ° C and were further analyzed by PCR. Only one, the larger clone, contained most of it of the 5 'end of the nucA gene. Sequencing of this cosmid clone was found by the nucA insert, that it starts at a Sau3AI site, 55 bases in from the beginning the mature sequence, and down continuously from the site; therefore he missed the signal sequence and added 55 bases from the 5'-end of the mature Gene.

PCR at Cosmid Library

Similarly, the packed phage was amplified from the cosmid library using 63K P1Rev (SEQ ID NO: 19) CTTAATTCCACAGCTTGTGAGC (where the 18th base may also be A and the 21st base also be T) and T3 (SEQ ID NO: 19) 7) or T7 (SEQ ID NO: 8) primer to extract the top sequence. Primer P1 Rev corresponds to the complement of nucleotides 319-341 of SEQ ID NO: 1 in the reverse direction. Reactions were also performed with the 4122ext (SEQ ID NO: 18) primer and the T3 or T7 primers. At 42 ° C, three blurred bands were seen at approximately 250, 400, and 550 bp, for the nucA P1 Rev / T3 or T7 pairs, all of which hybridized to the 420 bp probe. No tape was obtained with the 4122ext / T3 primer pair. Similar smeared bands were seen on the 4122ext / T7 primers. At 50 ° C, only the 250 bp band was obtained in all four reactions. T3 or T7 alone did not give band and the T3 / T7 primer pair gave very smeared, approximately 550 and 300 bp bands. The three smeared PCT products from the nucA P1 Rev / T7 and 4122ext / T7 PCR reactions were purified on a QIAquick ^™ column (Qiagen, Chatsworth, CA), eluted with 50 μl of water, and 2 μl of it was added to the pCR ^™ II vector cloned. Four of the six clones had inserts, with three different inserts for the nucA P1 Rev / T7 PCR product. However, only the approximately 350 bp insert was obtained with the 4122ext / T7 PCR product (seven out of eight had the insert). The sequence for the 4122ext / T7 clone corresponds to the nucA sequence, starting at the Sau3AI site, 55 bases herein from the 5 'end of the mature sequence, which was similar to the cosmid clone described above.

composition the complete nuc sequence

The full The nucA sequence became overlapping Clones composed of which 5 'upper Sequence, 3 'lower Sequence and the partial mature sequence described above contained. The complete nucA gene sequence is shown in SEQ ID NO: 1, nucleotides 229-2037. The signal peptide is encoded by nucleotides 229-303. The mature NucA protein is by nucleotides 304-2037 coded.

Example 3

Expression of NucA protein

The nucA gene was then cloned and expressed in E. coli either as a fusion protein or as a full-length NucA protein (with a signal peptide). The nucA gene was ligated into several E. coli expression vectors as listed in Table 1 and the cultures were appropriately induced for expression of the NucA protein. Whole cell lysates were normalized and run on SDS-PAGE gel and either stained with Coomassie or transferred to a nitrocellulose membrane and probed with rabbit anti-NucA polyclonal antibodies at a 1: 500 dilution in 5% BLOTTO. The clones are listed in Table 1 and were constructed as described below.

A fusion protein containing the NucA protein plus heterologous residues was expressed by cloning the nucA gene from plasmid pPX643 into the pRSET expression vector (see Figure 6). Plasmid pPX643 contains the complete mature nucA sequence cloned into pCR ^™ II vector as described in Example 2. The chosen version of pRSET was that with the reading frame labeled "C" by the manufacturer The vector contains the gene 10 T7 promoter, the ampicillin resistance gene, and has the ColE1 DNA replication origin.

pPX644 construction

plasmids pPX643 and pRSETC were digested with Asp718 restriction enzyme, by GENECLEAN method purified, digested again with XhoI enzyme, and the DNAs are electrophoretically treated on an agarose gel. The approximately 1.9 kb nucA-containing DNA fragment was isolated and ligated to the pRSETC isolated DNA. Which resulting clone was named pPX644. BL21 (DE3) pLysS transformants Called RZ1034.

The recombinant clone, pPX644, contains 51 additional amino acids, condensed to the N-terminal region of the mature nucA protein. These additional amino acids are provided by the vector and include six consecutive histidines just below the initial methionine, which help in the purification of the fusion protein. Whole cell extracts of induced cultures containing this plasmid were electrophoresed on an SDS-PAGE and a Western blot performed using rabbit polyclonal antisera against the NucA protein. Overnight cultures of RZ1034 isolates and BL21 (DE3) pLysS / pRSETC were inoculated into 10 ml of modified SOB (20 g tryptone, 5 g yeast, 0.6 g NaCl, 0.2 g KCl, adjusted to pH 7.0 with 5 N NaOH) plus ampicillin (100 μg / ml) in a 125 ml flask and grown at 37 ° C to a Klett Colorimeter reading of 126-155. One ml aliquot of uninduced cells was removed, centrifuged and resuspended with TE buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA) to the same volume in microliters (μl) as the Klett reading, ie a Klett Reading of 126 middle cells were resuspended using 126 μl of TE buffer. For induction, IPTG was added to the growing culture to a final concentration of 1mM and incubation continued for two hours, determined with a final Klett reading. One milliliter was removed and centrifuged in a 1.5 ml Eppendorf tube. The cell pellet was resuspended in TE buffer again based on its respective final Klett reading. Ninety μl of each non-induced and induced cells were removed to another Eppendorf tube, 30 μl of a 4x cracking buffer was added and the tube was placed in a boiling water bath for 10 minutes prior to running 20 μl on a 4-15% Gradient SDS-PAGE gel (Bio-Rad Laboratories) heated. The protein bands were transferred to nitrocellulose filter paper and a Western blot was performed using rabbit polyclonal NucA antisera and anti-rabbit Ig peroxidase (Tago Inc.). The bands were visualized using TMB reagent (Promega, Madison, WI). An anticipated 69 kD protein product was clearly detected in the blot, as in 7 shown. This confirmed the correct cloning of the nucA gene. In this example, the lack of observed inducibility of NucA was presumably due to the absence of the antibiotic chloramphenicol in the growth medium, which is required for stable maintenance of the pLysS plasmid (carrying the chloramphenicol resistance gene). Thus, loss of the pLysS plasmid would result in a high basal (non-induced) level of expression of NucA. In a separate experiment, the 69kD NucA protein was shown to be inducible in a Western blot when chloramphenicol was included in the growth medium (data not shown). Although the 69 kD protein band can be detected in a Western blot, it was not detected in a similar SDS-PAGE gel stained with Coomassie (data not shown). Without being bound by the following, one possible cause for non-detection of the recombinant protein in the Coomassie gel may be due to the 51 additional vector-encoded amino acid residues at the N-terminal end of the cloned nucA gene in pPX644.

One mature clone that displayed an expression level in a Coomassie gel was visualized using a slightly different fusion approach constructed. This clone, named pPX693, was as follows constructed:

pPX682 construction

The nucA gene was PCR amplified from P860295 genomic DNA using RSET-5 primer (SEQ ID NO: 15), which has the following sequence, the last 17 bases of which are reasonably nucleotides 304-320 of SEQ ID NO: 1 correspond (the primer was based on the pPX643 sequence generated using the degenerate primer P1):
RSET-5 GCTACGGCTAGCAAAGAAGCACCTCAAGC (SEQ ID NO: 15)
and the 63K RBam primer (SEQ ID NO: 13). (RSET-5 was designed to include a BamHI site at the 5 'end to facilitate transfer of the nucA fragment to the pRSET expression vector, see below). The amplified DNA fragment was cloned into the pCR ^™ II vector and designated pPX682. This construct encodes mature-length NucA.

pPX693 construction

The nucA BamHI fragment from pPX682 was ligated into the BamHI site of the expression vector pRSETC and the resulting plasmid was named pPX693. This yielded a clone expressing a fusion NucA with an additional 38 amino acids at its N-terminus. After cleavage with enterokinase, all but five amino acids were removed. The pRSETC vector also contains a polyHis region, which allows purification of the fusion protein over a nickel column prior to cleavage by enterokinase. The clone pPX693, when transformed into the E. coli BL21 (DE3) pLysS strain and induced with IPTG, showed a significant level of expression detectable on Coomassie stained gels. 8th shows that he was the major protein band in the cell lysate. However, after pulling a larger culture for protein purification, expression by Coomassie staining was undetectable. It was found that the plasmid was not kept stable in the expression strain. Subsequently, further clones with acceptable levels of expression were obtained so further work to stabilize pPX693 was not undertaken.

Native signal sequence

After the top sequence of clone pPX680 was obtained (an approximately 2 kb NsiI inverse PCR product cloned into pCR ^™ II vector as mentioned in Example 2), primers NdeF63K and NcoF63K were used to construct the native nucA gene of genome P860295 Full-length DNA amplification. The PCR product was cloned into two different expression vectors, pRSETC and pTrcHisC ( 9 ). These vectors have been maximized for expression using strong promoters that are inducible by IPTG. Plasmids pPX685, pPX688, pPX691 and pPX692, referred to in Table 1, were constructed as follows:

pPX685 construction

The full length nucA gene was cloned by PCR amplification from the genome of P860295. The primers used were NdeF63K and 63K RBam. Primer NdeF63K is SEQ ID NO: 1, nucleotides 229-255 with an NdeI site attached to the 5 'end for later transfer into pRSETC. The PCR product cloned in pCR ^™ II vector is called pPX685.

pPX692 construction

Subcloning the complete nucA sequence of pPX685 into pRSETC generated pPX692. Specifically, pPX685 was digested with NdeI and BamHI, the full-length nucA fragment was isolated and ligated into pRSETC, which had also been digested with NdeI and BamHI. The resulting plasmid was named pPX692. When pPX692 was transformed into the E. coli BL21 (DE3) pLysS strain and induced with 1 mM IPTG, a similar co-mass level of NucA protein was obtained (see 10 ) as compared to pPX693 (see 8th ).

pPX688 construction

The full length nuc A gene was cloned from genome P860295 DNA using PCR and primers NcoF63K and 63K RBam. Primer NcoF63K is SEQ ID NO: 1, nucleotides 229-255 with an NcoI site and a few extra bases for translational frame fitting attached to the 5 'end, especially bases ACCATGGGT for subsequent subcloning into pTrcHisC. The PCR product was cloned into the pCR ^™ II vector and the resulting plasmid was named pPX688. The NcoI linker added two amino acids to the N-terminal start region of the signal sequence.

pPX691 construction

Subclone the complete nucA sequence from pPX688 into pTrcHisC (see 9 ) generated pPX691. Specifically, pPX688 was digested with NcoI and BamHI, the full-length nucA fragment was isolated and then ligated into pTrcHisC, which had also been digested with NcoI and BamHI. When pPX691 was transformed into the E. coli InvαF 'strain and induced with 1 mM IPTG, a similar Coomassie level of NucA protein was obtained (see 10 ) as compared to pPX693 (see 8th ). The NcoI restriction site added two residues to the beginning of the native signal sequence that did not appear to affect the processing of the NucA signal sequence, as in 10 shown. The apparent molecular weight of the expressed protein appeared to be about 63 kD for a processed protein, rather than 66 kD for an unprocessed protein. The band at about 63 kD appeared to be the major protein band in the Coomassie stained gel. After purification and amino acid sequence analysis, it was shown to be processed. See Example 4 for protein sequencing.

The plasmid pPX691 was grown in a 2 L culture for purification together with pPX693 (fusion protein). However, pPX693 was found to be unstable in the BL21 (DE3) pLysS host while pPX691 was stably maintained in DH5α or InvαF 'hosts. Plasmid pPX691 was chosen as the clone of choice for protein purification of rNucA. A stability study for pPX692 was not performed because its expression was no better than pPX691. Please refer 10 ,

Even though the Haemophilus encoded nucA signal sequence shows good expression of rNucA in E. coli seemed as well as correct processing (ie removal of the signal sequence), were two commercially available E. coli expression vectors tries to find out which different signal sequences contained. PelB (pPX708 [made from pPX705] in pET20b vector, Novagen) and OmpT (pPX707 [prepared from pPX706] in pET12a vector, Novagen) were separated above the mature nucA gene as follows cloned:

PelB construction

The nucA gene was PCR amplified from pPX643 using primers 63K PelB (SEQ ID NO: 12) having the following sequence, the last 17 bases of which correspond approximately to nucleotides 304-320 of SEQ ID NO: 1 (primer based on the pPX643 sequence generated using the degenerate primer P1):
63K PelB GATATCAAAGAAGCTCCTCAAGC (SEQ ID NO: 12)
and 63K RBam (SEQ ID NO: 13) having the following sequence whose last 21 bases are the complement of nucleotides 2095-2115 of SEQ ID NO: 1 in the reverse direction:
63K RBam CGCGGATCCTGAAGTCTTCAAACCTAGGAC (SEQ ID NO: 13)

The approximately 1.8 kb nucA-containing PCR DNA band was gel isolated, cloned into pCR ^™ II, and the resulting plasmid was named pPX705.

pPX708 construction

Both Plasmids pPX705 and pET20b were digested with EcoRV and BamHI, DNAs were electrophoresed on an agarose gel and the correct DNA bands were isolated. The DNAs were ligated together and the resulting Plasmid was named pPX708. BL21 (DE3) pLysS transformants were Called RZ1070.

OmpT construction

The nucA gene was amplified from pPX643 using the primer 63K OmpT (SEQ ID NO: 14), which has the following sequence, the last 17 bases of which correspond approximately to nucleotides 304-320 of SEQ ID NO: 1 (the primer was based on the pPX643 sequence generated using the degenerate primer P1):
63K OmpT GGATCCAAAGAAGCTCCTCAAGC (SEQ ID NO: 14)
and 63K RBam (SEQ ID NO: 13). The approximately 1.8 kb nucA-containing PCR DNA band was gel isolated, cloned into pCR ^™ II, and the resulting plasmid was named pPX706.

pPX707 construction

pET12a Vector DNA was digested with BamHI enzyme, with calf-poor alkaline phosphatase (Boehringer Mannheim), electrophoretically on agarose gel treated and the DNA was isolated. The plasmid pPX706 was used with BamHI enzyme digested, electrophoresed on an agarose gel and that approximately 1.8 kb nucA-containing DNA fragment was isolated in gel. Either the pET12a, as well as pPX706 gel-isolated DNAs were combined ligated and the resulting plasmid was named pPX707. BL21 (DE3) pLysS Transformants were named RZ1065.

PelB-NucA & OmpT-NucA expression

One ml of overnight cultures of RZ1065 (pPX707: OmpT-nucA clone) and RZ1070 (pPX708: PelB-nucA clone) were each in 20 ml SOB plus chloramphenicol (30 μg / ml) plus ampicillin (100 μg / ml) in one 125 ml flask inoculated and grown at 37 ° C to OD ₆₀₀ = 1.0-1.3. Ten ml of the culture was transferred to another 125 ml flask and IPTG added to a final concentration of 1 mM. Both uninduced and induced cultures were grown at 37 ° C for an additional two hours. One ml of each of the cultures was removed and centrifuged in a 1.5 ml Eppendorf tube to pellet the cells. The supernatants from the induced cultures were preserved. The cell pellets were resuspended with 0.6-1.6 ml PBS to OD ₆₀₀ of approximately 1.0. One ml of the resuspended cells were recentrifuged and resuspended with 60 μl H ₂ O. To 60 μl of the resuspended cells or 60 μl of the supernatant was added 20 μl of a 4x cracking buffer and the tube was heated in a boiling water bath for five minutes. Twenty μl of each sample was electrophoresed on a 12% SDS-PAGE gel and stained with Coomassie Blue. Both the OmpT and PelB NucA-Fu protein were detected and were of the correct size for a processed protein ( 11 ). The yield of PelB NucA fusion protein expression was approximately 30% of the total cellular protein, consistent with the expression of full length NucA (with native signal, pPX691; 12 below) was comparable.

12 Figure 4 shows a comparison of expression of several signal sequence clones and also of a mature fusion clone with an optimized translation initiation region (TIR) sequence (11), pPX709. Clone pPX709 was constructed from clone pPX677 as follows:

pPX677 construction

Plasmid pRSETC was digested with NheI enzyme, extracted with phenol, precipitated with ethanol and digested again with NdeI enzyme. The DNA was electrophoresed on an agarose gel and the DNA band was gel-isolated. A NheI + NdeI linker was constructed by annealing the RSET.TIR upper oligo (SEQ ID NO: 10), which has the following sequence (which does not correspond to nucA):
RSET.TIR top TATGGCTATGTCTAACATGACTTACAAACATCATCATCATCATCATGG-TATGG (SEQ ID NO: 10)
to the RSET.TIR lower oligo (SEQ ID NO: 11), which has the following sequence (which does not correspond to nucA):
RSET.TIR below CTAGCCATRCCATGATGATGATGATGATGTTTGTAAGTCATGTTAGACA-TAGCCA (SEQ ID NO: 11)

This Linker was ligated to the NheI + NdeI digested pRSETC purified DNA and the resulting plasmid was named pPX677.

pPX709 construction

plasmid pPX677 was digested with BamHI, with calf-poor alkaline phosphatase (Boehringer Mannheim), electrophoresed on an agarose gel and the DNA was isolated. Plasmid pPX693 was digested with BamHI, electrophoresed on an agarose gel and the DNA became isolated. The isolated DNAs were ligated together and that resulting plasmid was named pPX709.

BL21 (DE3) pLysS transformants were named RZ1076.

The pPX691 (native signal sequence) clone and vector alone control were induced at OD ₆₀₀ of 0.8-0.9 with 1 mM IPTG for one hour. The pPX707 (OmpT signal), pPX708 (PelB signal) and pPX709 (TIR) clones were induced at an OD of about 1.0 for two hours. All cultures were normalized to an OD of about 1.0 and equal volumes loaded onto the gel. P860295 was grown for about 3.5 hours to an OD of about 1.6, then normalized to about 1.0. Induced pPX691 and induced pPX708 definitely showed an intense NucA band; in fact, it seemed to be the main protein band. However, pPX707 and pPX709 produced no apparent NucA-induced band. This shows that not all signal sequences or "optimized" expression vectors produce a high level of induced NucA protein.

Example 4

NucA protein purification and marking

Purification of nNucA protein from NTHi whole cell extract preparation

Whole bacterial cells (about 70 g wet weight of NTHi) were suspended in 200 ml of 0.1 M potassium phosphate (KPO ₄ ) / 3.0 M KSCN (pH 6.0) and stirred at room temperature for 60 minutes to give nNucA protein to extract. Cell debris was purified by centrifugation at 8000 rpm. using a Sorvall GS-3 rotor for 20 minutes at 4 ° C. The supernatant was collected and centrifuged at 60,000 rpm. using a Beckman 70Ti rotor for 60 minutes at 4 ° C further clarified. The supernatant was collected and dialyzed overnight at 4 ° C against 50 mM NaPO ₄ (pH 8.0).

column

The dialyzed whole cell extract was loaded onto a 25 ml ceramic hydroxyapatite (HA) column (80 μm Bio-Rad) equilibrated in 50 mM NaPO ₄ (pH 8.0). Bound protein was eluted with a 0.2 M NaPO ₄ step, followed by a linear NaPO ₄ gradient (0.2-0.5 M NaPO ₄ ). Fractions were screened for nNucA by SDS-PAGE and Western analysis, and positive fractions were pooled. The HA step was performed three times with three independent whole cell extract preparations. Native NucA from each of the three HA runs was pooled together and concentrated about 7-fold using an Amicon-stirred cell with a PM10 membrane. The HA concentrate was dialyzed overnight at 4 ° C against 2 L charges of 50 mM Tris-HCl / 1 mM EDTA (pH 9.0). The material was loaded on a MonoQ HR5 / 5 column (Pharmacia) equilibrated in 50 mM Tris-HCl / 1 mM EDTA (pH 9.0). Bound protein was eluted with a linear NaCl gradient (0-1.0 M NaCl in 50 mM Tris-HCl / 1 mM EDTA (pH 9.0)). Fractions were screened for nNucA by SDS-PAGE and pooled. A total of 2.5 mg of purified nNucA was isolated from about 70 g wet weight of NTHi cells. NucA native protein, purified using this protocol, had a single band on an SDS-PAGE gel, corresponding to a molecular mass of about 63,000 daltons.

NaCl Extraction from nNucA

The following extraction protocol for nNucA protein was used as an alternative to extraction by KSCN. Bacterial cells (about 37.5 g wet weight of NTHi) were suspended in 150 ml of 50 mM NaPO ₄ (pH 7.0) and disrupted by three passages through a French pressure cell at 1000 psi. Cell debris and membranes were centrifuged at 55,000 rpm. pelleted using a Beckman 70Ti rotor for 20 minutes at 4 ° C. The pellet was resuspended in 132 ml of 10 mM HEPES / 1.0 M NaCl (pH 7.5) and stirred at 4 ° C for 20 minutes to extract the nNucA protein. Cell debris and membranes were centrifuged at 60,000 rpm. using a Beckman 70Ti rotor for 60 minutes at 4 ° C. The supernatant was collected and dialyzed overnight at 4 ° C against 50 mM NaPO ₄ (pH 8.0). Subsequent HA and MonoQ chromatographic steps were performed as described above for the native NucA protein. Yields of nNucA using NaCl extraction were comparable to those obtained by the other method described above.

cleaning of rNucA protein

Raw extract preparation

One Method using cell lysis by passage through a French pressure cell and two chromatography steps were for the purification of rNucA protein developed. Bacterial cells expressing rNucA (about 30 g wet weight of E. coli InvαF '/ pPX691) were analyzed in 80 ml of 50 mM Tris-HCl / 1 mM EDTA (pH 8.0) and suspended by three Passages through a French pressure cell torn at 1000 psi. Cell debris and membranes were removed by centrifugation at 55000 rpm. using a Beckman 70Ti rotor for 20 minutes at 4 ° C away. The supernatant was collected and over Night at 4 ° C against two 4 l changes of 50 mM Tris-HCl / 1 mM EDTA (pH 8.0) dialyzed.

column

The dialyzed crude extract supernatant was loaded on a 50 ml Trimethylaminoethyl (TMAE) Fractogel ^™ anion separation column (EM Separations Technology) equilibrated in 50 mM Tris-HCl / 1 mM EDTA (pH 8.0) at room temperature. Most of the contaminating proteins bound to the column. The column flow containing rNucA protein as well as several contaminant proteins was collected and dialysed overnight at 4 ° C against 20 mM HEPES / 1 mM EDTA (pH 7.0). The material was then loaded onto a MonoS HR10 / 10 cation exchange column (Pharmacia) equilibrated in 20 mM HEPES / 1 mM EDTA (pH 7.0), which binds the NucA protein. Bound protein was eluted with a linear NaCl gradient (0-1.0 M NaCl in 20 mM HEPES / 1 mM EDTA (pH 7.0)). Most of the rNucA eluted at about 0.4 M NaCl. Fractions were screened for rNucA by SDS-PAGE and pooled. A total of 80 mg of purified rNucA was isolated from about 30 g wet weight of cells. The rNucA protein purified using this protocol had a single band on an SDS-PAGE gel corresponding to a molecular mass of about 63,000 daltons.

Amino acid sequence analysis

For amino acid sequence analysis of the native protein were about 250 μl of the sample, which was 100 μg purified Protein precipitated with 90% ethanol for 30 minutes at 0 ° C followed of centrifugation in a microcentrifuge. The pellet was in 20% acetic acid resuspended and subjected to amino-terminal amino acid sequence analysis. For the recombinant protein was 80 μl the sample containing 80 μg rNucA to a ProSpin cartridge (Applied Biosystems, Foster City, CA) and centrifuged. The polyvinylidene difluoride (PVDF) Mem bran containing the sample was removed and washed with Washed 20% methanol. The membrane was amino-terminal amino acid sequence analysis subjected.

Amino-terminal Amino acid sequence analysis was performed using an Applied Biosystems Model 477A protein / peptide Sequencer equipped with an on-line Model 120A PTH Analyzer (Applied Biosystems). After cleavage of each consecutive amino-terminus the anilinothiazolinone derivative formed became more stable Phenylthiohydantione (PTH) derivative by treatment with 25% trifluoroacetic acid (TFA) at 64 ° C for 20 Minutes converted. The PTH derivatives were separated and on the PTH analyzer by reverse phase HPLC using a Brownlee PTH C-18 Column (particle size 5 mm, 2.1 mm internal diameter × 22 cm length; Applied Biosystems) with a two-solvent gradient system according to the instructions identified by the manufacturer.

For the native Protein became unique the first 26 residues (SEQ ID NO: 2, residues 26-51) identified, with the exception of one additional unidentified Peaks at residue 1. For the recombinant protein was 18 of the first 19 residues (SEQ ID NO. 2, residues 26-44) clearly identified. Both residues 1 and 2 contained an additional unidentified peak and residue 13 (SEQ ID NO: 2, residue 38) can not be identified. Simultaneity of the amino-terminal Sequence of the recombinant NucA protein with that of the native protein showed identical post-translational processing of the signal sequence in Protein expressed in E. coli with that in Haemophilus.

Example 5

Nukleotidasewirskamkeit of NucA protein

ectoenzymes are called intrinsic membrane proteins with their catalytic Places at the extracellular surface the membrane defined (12). One class of ectoenzymes is ecto-phosphohydrolase (5'-nucleotidase). It gives many reports of both bacterial and mammalian 5'-nucleotidases, which each show different substrate reactivities. mammal 5'-nucleotidase enzyme uses AMP (adenosine monophosphate nucleotide) as a substrate and produces A (adenosine nucleoside) and P (phosphate), while Bacterial enzymes ATP (Adenosine triphosphate nucleotide), ADP (Adenosine diphosphate nucleotide) and AMP, as well as other nucleotides.

one the early one Reports that the E. coli 5'-nucleotidase protein investigate, suggesting that it is in the periplasmic space the cellular Membrane was and that this protein was unique, because it has both sugar hydrolase and mononucleotide activity (13; 14). A later one Study by Burns and Beacham (15) showed a nucleotide sequence analysis and N-terminal protein sequence of the mature E. coli 5'-nucleotidase gene ushA. Based on the protein sequence and the DNA gene sequence contains the UshA protein a signal sequence which proteolytically removes in the mature protein becomes. The anticipated molecular weight of the mature protein is 58 kD.

marine Luminescent Vibrio and Photobacterium strains contain a membrane-bound specific 5'-nucleotidase enzyme (16). The halophilic marine bacterium Vibrio parahaemolyticus encodes for a 5'-nucleotidase gene with the name nutA (17). The nutA gene is cloned, sequenced and expressed in E. coli. The 5'-nucleotidase enzyme can be a lipoprotein, inserted into the outer membrane be. 5'-Nukleotidasewirksamkeit is also detected in gram-positive bacterium Bacillus subtilis been (18).

amino acid comparison

An amino acid comparison of rat liver 5'-nucleotidase, E. coli 5'-nucleotidase (UshA), and 2 ', 3'-cyclic phosphodiesterase revealed a region of striking homology, indicating that this domain was involved in catalytic activity and / or binding may be involved by substrate (19). Further comparison of different 5'-nucleotidase sequences, rat, human, UshA, NutA and NucA revealed two very highly homologous regions. These regions span amino acids 119-152 of the NucA sequence of SEQ ID NO: 2. For example, complete homology of the NucA amino acid sequence with the human nucleotidase amino acid sequence is present at residues 124-130 and 148-151 of SEQ ID NO: 2. Although the overall degree of homology between NucA and human nucleotidase proteins is very small and limited primarily to those regions described above having four or more contiguous homologous amino acids, deletion of either or both of these regions should diminish any potential immune cross-reactivity of a vaccine contains the NucA protein.

5'-nucleoside Delivery Test

A Protein search was performed by the NCBI BLAST e-mail service the nucA gene sequence performed. The search pointed to somewhat limited amino acid homology with known ones Transverse 5'-nucleotidase precursors through species, ie humans, rats and E. coli. A Lipman-Pearson Protein alignment (DNAStars) was determined by comparing NucA protein with rat nucleotidase protein. A 33.0 similarity index was determined which NucA (amino acids 36-333) with rat nucleotidase (Amino acids 30-329) compared. An initial one 5'-nucleoside Delivery Test using rat 5'-nucleotidase reaction conditions as described by Ikehara et al. described (20) performed to to determine if the NucA protein possesses 5'-nucleotidase activity.

One 5'-nucleotide like AMP is a polar substrate which is not attached to a TLC plate will migrate if a solvent as methanol / chloroform is added. A nucleotidase will become split off from the phosphate group and leave the adenosine, which is less polar. Adenosine is present on the plate migrate from methanol / chloroform. So a TLC spot means which one migrated that the AMP has been cleaved by a nucleotidase is.

One-tenth of one ml of NTHi P860295 cells was centrifuged, washed with PBS buffer and resuspended with 10 μl of PBS. Purified rNucA and nNucA protein concentrations were at 200 μg / ml. 5 'nucleotidase reactions (100 μl) were performed in 1.5 ml Eppendorf tubes containing 0.1 M Tris-HCl, pH 7.5, 5 mM MgCl ₂ , 0.1 M KCl, 5 mM AMP with or without added cells or protein. Each tube was incubated at 37 ° C for 60 minutes. Two μl of the reaction was spotted on a silica TLC fluorescent plate and eluted with 20% methanol in chloroform. The plate was then viewed using a short wave 254 nm UV source. As in 13 both the recombinant and native purified NucA protein and P860295 cells exhibited 5'-nucleotidase activity.

Phosphate Delivery Test

In a more quantitative 5-nucleotidase assay, inorganic phosphatase released upon hydrolysis of 5'-nucleotides was determined colorimetrically using a modification of the method of Chen et al. (21) determined. Reaction mixtures contained 100 mM Tris-HCl (pH 9.0), 6.6 mM MgCl ₂ , 1.65 mM 5'-AMP (or other nucleotide substrate) and enzyme in a final volume of 750 μl. The reaction mixture was incubated at 37 ° C for 30 minutes and the reaction stopped with the addition of 250 μl of 20% TCA. Controls were run for each reaction in which enzyme was added after the addition of 20% TCA. Precipitated protein was removed by centrifugation and 200 μl of supernatant was withdrawn into a fresh test tube. One hundred microliters of 1.0 N HCl was added, followed by the addition of 750 μl of ammonium molybdate reagent (3.0 ml of 10% ascorbic acid plus 18 ml of 3, 4 mM ammonium molybdate in 1 NH ₂ SO ₄ ), and the mixture became 37 ° C for 30 minutes. After allowing the tubes to cool to room temperature, the absorbance at 650 nm was determined. One unit of 5'-nucleotidase activity is defined as the amount of enzyme which gives an absorbance change of 1.0 at 650 nm min ^-1 at 37 ° C.

PH Optimum

Of the optimal pH for the hydrolysis of 5'-AMP was found between 8.5 and 9.0 (data not shown); however, pHs were over 9.0 not studied.

Effect of Mg ⁺⁺

Divalent cations, particularly Mg ⁺⁺ , have been shown to stimulate or, in some cases, to be an absolute requirement for 5'-nucleotidase activity in both prokaryotic and eukaryotic systems (16). The rNucA protein was also shown to be stimulated by the addition of MgCl ₂ . The addition of up to 6.6 mM MgCl ₂ gives approximately 2-fold stimulation of enzymatic activity (data not shown).

Substrate specificity

From the rNucA 5'-nucleotidase has been shown to be a relatively broad substrate-specific Profile. The enzyme has been shown to be a variety at 5'-mono-, di- and triphosphate nucleotides in addition hydrolyzed to 5'-AMP. For all nucleotides studied, the monophosphate nucleotide appeared to be the preferred substrate, except in the case of uridine, where UTP across from UMP or UDP was preferred. The rNucA 5'-nucleotidase appeared specific for 5'-nucleotides because it did not show efficacy in 3'-AMP (data not shown).

Phosphatase Test

One Phosphatase test was performed to determine if the NucA protein is non-specific hydrolyzing Effectiveness in addition to its specific 5'-nucleotidase activity has. Phosphatase was synthesized using p-nitrophenyl phosphate (PNPP) tested as a substrate. Reaction mixtures (final volume 750 μl) contained 100mM Tris-HCl (pH 9.0), 25mM PNPP and rNucA. The reaction mixture was at 37 ° C for 30 Incubated for minutes and the reaction by placing the tubes on Ice stopped. The absorbance at 410 nm was determined. recombinant NucA had no detectable potency in PNPP (data Not shown). This indicates that NucA is a nucleotidase without phosphatase activity. In contrast, E. coli UshA Phosphatase activity in addition to its known nucleotidase activity (13; 14).

Example 6

Monoclonal antibody inhibition of rNucA 5'-nucleotidase

Monoclonal antibody (MAb) Nt63-34-25 grown to purified nNucA protein as shown in Example 7 below was tested for its ability to inhibit and / or precipitate the enzymatic activity of rNucA. MAb Nt63-34-25 has been shown to cross-react with rNucA as well as nNucA (data not shown). Inhibition or precipitation of enzyme activity by the anti-nNucA MAb would confirm that the rNucA protein is indeed a 5'-nucleotidase. Increasing concentrations of MAb (0, 2.5, 5, 10 and 20 μl) were incubated overnight at 4 ° C with purified rNucA protein. An identical sequence was set up which also contained 50 μl of protein A beads (Pierce) in addition to rNucA protein and MAb to facilitate immunoprecipitation. The mixtures were centrifuged and the supernatants were subsequently tested for 5'-nucleotidase activity using the phosphate release assay as described above. Percent efficacy relative to the control which did not contain MAb was calculated. 14 shows that 5'-nucleotidase activity was inhibited when the concentration of MAb Nt63-34-25 was increased. The addition of 10 μl of MAb gave about 25% inhibition of enzyme activity, while the addition of 20 μl had about 45% inhibition. The effect of MAb is enhanced if protein A beads are added to facilitate immunoprecipitation of the rNucA protein. In the presence of protein A beads, the addition of 10 μl of MAb resulted in about a 60% decrease in 5'-nucleotidase activity, while the addition of 20 μl had about 80% inhibition. Complete inhibition of 5'-nucleotidase activity was achieved with higher concentrations of MAb NT63-34-25 (data not shown). These data confirm that the rNucA protein is indeed a 5'-nucleotidase.

Example 7

Generation of antibodies and Animal studies-Preparation of hybridomas for monoclonal antibodies

Purified NucA protein preparations as described above were used in immunizing mice to produce monoclonal antibodies. Female, eight week old BALB / c mice were intraperitoneally three times over a four week period (week 0, 2, 4) with 5 μg NucA protein purified from NTHi strain P860295 and 25 μg of MPL ^™ adjuvant in 0 , 1 ml dose inoculated. Two separate fusion procedures were performed. The first fusion occurred after a resting period of three months using an intraperitoneal (IP) enhancer dose of 0.5 μg NucA protein; the second fusion took place in the same way after a rest period of four months using an IP amplifier dose of 5 μg NucA protein. During immunization and resting period, mouse sera were obtained (week 0, 6) and tested for antibody efficacy by ELISA using NucA protein as the applying antigen.

For both Fusions were made to the spleen of two immunized mice about 72 hours after the won last injection and was treated with non-secreting X63Ag8.653 Mouse (BALB / c) myeloma cells in 7: 1 or 5: 1 ratios (Splenocytes: myeloma) respectively for combined first and second merger. The cells were left for four minutes in 50% (w / w) polyethylene glycol 1500 and 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (D-MEM) fused. The fused cells were in selection medium, D-MEM by hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum and 10% NCTC-109 media supplement (Gibco-BRL). The merger efficiencies (wells with colony growth versus number of seeded Troughs) were 26.4% (119/450), or 76.4% (346/450). The reactivity was by SDS-PAGE Western Blot, dot blots and ELISAs using of the NucA protein and whole cells. Positive reactors were identified as 1-25 denoted as Nt63 (first fusion) or Nt63 / 2 (second fusion) and for further identification kept.

Selected hybridomas of interest were subcloned once by limiting dilution procedure. Monoclonal antibodies were used as tissue culture fluid supernatant (TCS) provided by 50% saturated Ammonium sulfate precipitation (SAS-TCS) or ascites concentrated.

generation of polyclonal serums

Polyclonal sera were generated in New Zealand white rabbits. Six rabbits were screened for background titers using P860295 whole cell ELISA. Two rabbits with the lowest titers were selected for immunization with nNucA protein at 25 μg per dose plus 25 μg MPL ^™ in saline at weeks 0, 4 and 8. The rabbits were bled at week 10. Protein was purified as described above and run on a 12% SDS-PAGE gel to increase the purity of its approximately 90% degree. The 63 kD band was excised from the coomasie-stained gel and passed in succession through 18G, 20G and 22G needles. The adjuvant MPL ^™ in 0.9% saline was added and 0.2 ml was injected into rabbits. The polyclonal sera gave high background in colony blots, even after absorption on XL1-Blue MRF ', Y1090R', Y1089R 'and Y1088 cells.

immunogenicity

immunogenicity, carried out nNucA and rNucA are summarized in Tables 2-6 below. pooled Sera were re Antibody titers to nNucA and / or rNucA, whole cell titers to several heterologous NTHi strains and also bactericidal titers to mainly P861454 tested.

table Figure 2 illustrates the dosages used in the immunogenicity studies were used.

table 3 presents a compilation of whole cell (WC) ELISA data Studies Nos. 1 and 3.

table Figure 4 depicts WC ELISA data from study # 2, including subtyping the IgG antibody class.

table Figure 5 illustrates bactericidal data from Studies Nos. 1, 2 and 3. Bactericides Titers for Study # 3 was an average of two tests, except for group N755. Immunogenicity # 1 showed that the nNucA protein has the potential to be a vaccine candidate to be. See Tables 3 and 5. It showed elevated whole cell titers to all heterologous strains, which were tested at the time. More importantly it showed bactericidal Effectiveness for two out of four heterologous strains tested (Table 5). Studies No. 2 and 3 repeated the whole cell data. See Tables 3 and 4. Bacterial activity of these sera was heterologous for two strains shown (data for Strain P861454 are shown in Table 5). Three more strains were tried, but due to technical difficulties could not Data are reported.

table 6 represents antibodies ELISA titers from these three studies.

Table 4 Whole cell ELISA on week 6 sera Study # 2 ELISA for IgG endpoint = 0.1

used Standard (std) were pooled week 8 mouse sera from E951, immunized with total OMP from P8602955.

IgG subtype for TN106

The Results of Tables 2-6 presented data will now be presented.

Study No. 1

Study # 1 was performed with nNucA protein, isolated and purified as described in Example 1. Four groups of Swiss-Webster mice, five mice per group, were immunized with either 1, 5, 10 or 20 μg nNucA per mouse at weeks 0, 4, 6 and 8. The adjuvant used was MPL ^™ , 50 μg per dose. Antibodies to nNucA and whole cell titers to the homologous P860295 NTHi strain were obtained on pooled sera from all bleeds. Data for weeks 0 and 8 are shown (Tables 3 and 6). Whole cell titers to heterologous strains and bactericidal titers were obtained on group E548, the 5 μg dose group giving the highest whole cell titers to the homologous P860295 strain at both weeks 6 (data not shown) and 8 (Table 3). After the second immunization, both whole cell and purified protein ELISA titers were enhanced compared to the initial bleeding (data not shown). Two- to three-fold enhancement was also seen after the third immunization, except for the 10 μg dose, which showed a 13-fold increase in ELISA titer. The nNucA protein elicited high whole-cell ELISA titers against eight heterologous NTHi clinical isolates (Table 3) and demonstrated conserved, surface-exposed epitopes. From this study, 5 μg was chosen as the preferred immunization dose for further experiments. A bactericidal assay was performed using sera from the 5 μg dose (E548) on four heterologous NTHi strains. A significant bactericidal titer is considered a four-fold increase over pre-immunoads. Significant bactericidal activity was demonstrated over the homologous P860295 strain as well as for the heterologous NTHi isolates P810384 and N830161E. The titer against strain P861454 may be significant, but could not be determined in this assay because the lowest dilution tested was 1: 5 and the pre-immune sera had no efficacy at that level (Table 5).

Study No. 2

Study No. 2 was performed to compare the immunogenicity of nNucA with rNucA, both with and without adjuvants. Two adjuvants were compared: MPL ^™ and Stimulon ^™ QS-21. Five Balb / c mice per group were used for these studies. The NucA proteins used for these studies were purified as described in Example 4 with the nNucA protein KSCN extracted from P860295. Mice were immunized at weeks 0 and 4 and bleed at weeks 0, 4 and 6. Sera from week 0 and 6 bleeds were analyzed for ELISA-reactive antibodies to both purified nNucA, rNucA and whole NTHi cells.

The results are shown in Tables 4 and 6. The nNucA and rNucA proteins were not highly immunogenic in the absence of excipients. Both proteins elicited high ELISA titers against themselves or against the other form of the protein when used with either adjuvant (Table 6). No difference was detected when either nNucA or rNucA was used as the immunizing antigen and the sera reacted to each protein. It thus appears that the rNucA elicits antibodies indistinguishable from those elicited by nNucA. Whole cell ELISA titres for these antisera are shown in Table 4. A total of 16 H. influenzae clinical isolates were examined using these antisera. Only those groups containing excipients elicited full-cell high titer reactive antisera. The antisera were widely cross-reactive across the strains, demonstrating that both purified native and recombinant NucA retained surface-exposed epitopes that elicit antibodies when injected into mice. No significant differences were observed between either adjuvant or protein combination in the ability to elicit surface-reactive antibodies. Non-excipient groups showed background levels of surface-reactive antibodies. The subclass distribution of the antibody response to an isolate was determined and the results shown in Table 4. The MPL ^™ groups appear to have a mixed IgG1, IgG2a / b response, while the Stimulon ^™ QS-21-assisted native protein was a major IgG1 response. In contrast, the Stimulon ^™ QS-21 rNucA group was a mixed IgG1, IgG2a / b response. However, these answers are only for one try and may not represent the expected subclass profile for these adjuvants in additional trials.

Study No. 3

Study # 3 was performed to look at rNucA dose response when formulated at 50 μg MPL ^™ per dose and to collect sera for the protection study shown in Table 7 below. The rNucA and nNucA were the same preparations as for study # 2. Three groups of 10 mice each were treated with rNucA at 1 μg per dose (group N710), 5 μg (group N711) or 10 μg (group N71). An additional group of 20 mice, N755 (approximately 10 weeks old instead of 6-8 weeks) was later added to the study to provide extra sera for the protection study. A fifth group of 10 mice, N713, was immunized with 5 μg nNucA. All doses contained 50 μg of MPL ^™ per dose and mice were immunized at weeks 0, 4 and 8. Blood was collected at weeks 0, 4, 6 (N755 only), 8 and 10. See Table 2 for dosages.

The primary Answer, as indicated by the IgG ELISA titer for week 4 sera (data not shown) is affected by the dose of rNucA. Of the lowest titer was obtained with the lowest rNucA dose group. A two to three times higher Titre was from the 5 μg Dose group received. The titer for the 10 μg dose group was again about twice that for the 5 μg Dose. Similar Titers were for both the nNucA and rNucA groups were obtained at 5 μg doses. N755 resulted the highest primary Answer, but the lowest gain, about 45 times after the second dose. All other groups increased about 200-fold after second dose. Groups N710 and N755 increased about two to three times after the third dose; Groups N711, N712 and N713 actually fell easily from. All titers were comparable after three immunizations. Please refer Table 6.

All strains at a 10 μg Dose gave a small primary WC answer. DL208 and P861454 were the only two strains that a small primary Answer to all groups resulted. All titers were after the second and third immunizations comparable. N755 gave a higher primary response for DL208, P810384 and P861454 (which also has a high background but the titers were again after the second and third Immunization comparable. However, bleeding showed at week 6 higher and with some tribes the highest Titer opposite Weeks 8 and 10 for Group N755. See Table 3 for results from weeks 0 and 10.

Here the results repeated again those of previous studies, which the cross-reactivity opposite to the sera all strains tested showed, even opposite the encapsulated type b Eagan tribe. However, it became a definite one Titers for Eagan not received, possibly due to the presence of the capsule, what accessibility of the antigen on the cell surface may have inhibited.

Bactericidal activity of study # 2 antisera was determined against two NTHi strains, TN106 and P861454, both heterologous isolates relative to the source of the nucA gene. All sera except the Stimulon ^™ QS-21 rNucA group showed bactericidal activity against TN106. Results of the BC test against P861454 are shown in Table 5. The only groups that showed a significant (> 4-fold increase) bactericidal titer over P861451 were the MPL ^™ groups N091 and N087. Thus, either native or recombinant NucA protein, aided by MPL ^™ , was able to elicit a broadly cross-reactive whole cell ELISA response, high ELISA titres against any of the proteins and bactericidal activity against heterologous NTHi isolates.

Rattenjunge protection study

Four Day old Sprague-Dawley rats were randomized into 10 groups with one Mother divided into each group of 10 boys. The boys were immunized intraperitoneally (IP) as shown in Table 7.

• 1 Geometrische Mittel der Titer (GMTs) basierten auf gleicher Gewichtung an allen zählbaren Platten.
• 2 GMTs basierten auf höherer Gewichtung auf zählbaren niedrigeren Verdünnungen. Zählbare Platten waren < 500 cfu/Platte. Die Nachweisgrenze betrug 100 cfu/ml mit Plattieren von 10 μl der Verdünnung.

Groups P174 and P175 received rabbit anti-sera immunized with a 16kD NTHi protein designated P6 (also known as HiPAL or PBOMP-1 (22)). Group P176 received a monoclonal antibody raised against NTHi polyribosyl ribitol phosphate (PRP). Group P177 received PCM buffer (10 mM NaPO ₄ , pH 7.4, 150 mM NaCl 0.5 mM MgCl ₂ , 0.15 mM CaCl ₂ ) as a buffer control. All dilutions of sera and cells were performed in PCM buffer. About 23 hours later, they were challenged IP with 49.5 organisms (0.1 ml) of virulent H. influenzae type b, Eagan strain. Then, 20-24 hours after challenge, the rat cubs were bled and plated for bacterial counts. The tails were cut and 10 μl of blood were picked up with a P20 Rainin Pipetman and diluted in 90 μl of PCM buffer at RT. Dilutions were vortexed and held at 4 ° C until further dilutions were prepared and 10 μl of each dilution was plated twice on chocolate agar. Plates were incubated in 5% CO ₂ incubator at 36.5 ° C overnight. The results of the protection study are shown in Table 8:

• 1 Geometric means of titers (GMTs) were based on equal weighting on all countable plates.
• 2 GMTs were based on higher weighting at countable lower dilutions. Countable plates were <500 cfu / plate. The detection limit was 100 cfu / ml with plating of 10 μl of the dilution.

plates for groups P175 and P176 were 0 at the lowest dilution and can be anywhere from 0-99 cfu / ml be. Here was a number, 50 cfu / ml for the purpose of the calculation of assigned to geometric mean (GMT).

Plates that were too numerous to count, even at the highest dilution, were also numbers based on
Estimates assigned:> = 1000 cfu / plate
>> = 2000 cfu / plate
Lawn = 6000 cfu / plate

p-value: Kruskal-Wallis is a conservative p-value test that uses is when sample values a large Cover bandwidth. He is not that sensitive to much huge Values. P values are <0.05 statistically significant in this test.

The Rat young animal model for Hib meningitis shows the ability of antibodies, bacteremia (and subsequently death) due to Hib decrease. rabbit in general, give some non-specific immunity to Hib in the blood of rat cubs, as can be seen when the week 0 cfus of the mouse and rabbit antisera. The model thus uses pre-immune rabbit sera as a negative control for the immune rabbit sera. As expected, rabbits reduced control antisera across from the GMP of Hib in the blood of rats from 300 to 0.5 P6 protein and thus showed reduced bacteremia, as did the mouse monoclonal antibody across from the PRP capsule of Hib (group P176). Mouse anti-rNucA sera a dilution of 1: 2 significantly reduce the levels of bacteremia compared with week 0 (pre-immune) sera and showed a positive result in this animal model and the ability to encapsulated hib to oppose. These results show the effectiveness the anti-rNucA sera in this model and its vaccine potential across from Haemophilus influenzae.

Example 8

Specificity of the nucA 420 bp probe

The 420 bp DNA probe described above was tested for specificity in identifying Haemophilus species. The probe was used in Southern hybridizations to generate a partial restriction map of the nucA gene and in dot-blot hybridization to identify the Haemophilus species. The cultures were grown to OD ₆₀₀ of about 1.0 and stored at -20 ° C. Five μl culture was spotted onto a dry Hybond-N membrane with a 3 mm Whatman paper underneath to dab off excess liquid. The membrane was air dried for about 10 minutes. It was lysed in 10% SDS, then denatured in denaturing buffer (1.5 M NaCl, 0.5 M NaOH) for 7 minutes at RT. Excess buffer was blotted with 3 mm Whatman paper. The membrane was neutralized in neutralization buffer (1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2, 1 mM EDTA) for 3 minutes at RT. The membrane was placed on 3 mm Whatman paper to blot off excess buffer and the neutralization treatment was repeated. The membrane was rinsed in 2X SSC and air dried on 3mm Whatman paper. The DNA was cross-linked to the membrane using a Stratagene UV cross-linker in auto mode. The membrane was prehybridized in prehybridization buffer (5x SSC, 1.0% blocking reagent, 0.1% N-lauroyl sarcosine, 0.02% SDS) at 65 ° C for 2-4 hours. Hybridization was performed in prehybridization buffer plus probe (PCR dig-dUTP labeled P860295 chromosomal DNA with specific primers yielding the same 420 bp fragment as described above) at 65 ° C overnight. Two washes were performed in 2 x SSC-0.1% SDS at 65 ° C for 5-15 minutes. Another two washes were performed in 0.1x SSC-0.1% SDS at 65 ° C for 15 minutes. The membrane was developed according to the Genius Kit protocol (Boehringer Mannheim).

• 1. E. coli InvαF'
• 2. pTrcHisC in InvαF'
• 3. pPX691 in InvαF'
• 4. NTHi P810384
• 5. NTHi P810568
• 6. NTHi P860295
• 7. NTHi 861454
• 8. NTHi P880859
• 9. NTHi DL208
• 10. NTHi H305
• 11. NTHi N1955
• 12. NTHi N830161E
• 13. NTHi SH1013
• 14. NTHi SH1014
• 15. NTHi SH1015
• 16. NTHi TN106
• 17. Hib Eagan
• 18. GC LB2
• 19. GC Pgh3-2
• 20. H. pylori
• 21. M. catarrhalis 035e
• 22. S. typhimurium 3261

Listed below are the types and strains of cell cultures that were spotted for point hybridization with this nucA probe. All Haemophilus strains were visibly positive, including Tyb b, Eagan strain. The only non-Haemophilus species that was positive was the E. coli culture containing the rnucA plasmid pPX691. All other species were negative for this probe. Please refer 15 ,

• 1. E. coli InvαF '
• 2. pTrcHisC in InvαF '
• 3. pPX691 in InvαF '
• 4. NTHi P810384
• 5. NTHi P810568
• 6. NTHi P860295
• 7. NTHi 861454
• 8. NTHi P880859
• 9. NTHi DL208
• 10. NTHi H305
• 11. NTHi N1955
• 12. NTHi N830161E
• 13. NTHi SH1013
• 14. NTHi SH1014
• 15. NTHi SH1015
• 16. NTHi TN106
• 17. Hib Eagan
• 18. GC LB2
• 19. GC Pgh3-2
• 20. H. pylori
• 21. M. catarrhalis 035e
• 22. S. typhimurium 3261

GC for 18. and 19th is Neisseria gonorrhoeae. These results showed the specificity the probe for nucA sequences.

Example 9

Preservation of the NucA sequence under H. influenza

Whole cell lysates of several NTHi strains and type b, Eagan strain, were analyzed on Westerns probed with Mab Nt63-34-25 or rabbit polyclonal, both to nNucA. All strains tested showed a retained 63 kD band (size on an SDS-PAGE gel) with the nNucA antisera (data not shown). Several NTHi strains and type b, Eagan and Whittier strains were also grown to OD ₄₉₀ of about 1.0, diluted with 0.4% formaldehyde fixed to OD ₆₂₀ of about 0.2 in PBS, and dried for WC ELISA pulled on plates. See Tables 3 and 4. All strains tested showed an increased titer on the WC ELISA.

As described above was the sequence of the NucA protein of strain P860295 (SEQ ID NO: 2). Next the nucA fragment (with or without signal sequence) became eight others NTHi genomes and type b, Eagan strain using primers amplified, prepared by sequencing the nucA region from P860295 Genome.

The PCR fragments were cloned into pCR ^™ II and sequenced. Full sequences of the mature nucA gene region were obtained for strains P810384, P810568, P861454, P880859, N1955, TN106, SH1014, SH1015 and Eagan. Protein assembly was generated from the deduced amino acid sequences of the mature NucA protein including that of P860295. Four of the strains (P880859, TN106, SH1015 and Eagan) are 100% identical to P860295. The other strains differ from P860295 by one to seven amino acid residues (see Table 9). This demonstrates the considerable extent to which the nucA gene is conserved among the tested H. influenzae strains.

Table 9 Mature NucA amino acid residue differences between strain P860295 and other H. influenzae strains

bibliography

• 1. Moxon, E.R., J. Antimicrob. Chemother., 18 Supp. A, 17-24 1986).
• 2. Murphy, T.F., and Apicella, M.A., Rev. Infect. Dis., 9, 1-15 (1987).
• 3. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
• 4. Barenkamp, S.J., et al., Infect. Immun., 52, 572-578 (1986).
• 5. Moxon, E.R., et al., J. Infect. Dis., 136, 5186-190 (1977).
• 6. Lam, J.S., et al., Current Microbiol., 3, 359-364 (1980).
• 7. Ulmer, J.B., et al., Science, 259, 1745-1749 (1993).
• 8. Fynan, E.F., et al., Proc. Nat'l. Acad. Sci., USA 90 11478-11482 (1993).
• 9. Silhavy, T., et al., Experiments with Gene Fusion pages 95-96, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984).
• 10. Faure, D., BioTechniques, 13, 22-26 (1992).
• 11. Sprengart, M.L., et al., Nuc. Acids Res., 18, 1719-1723 (1990).
• 12. Luzio, J.P., et al., Pages 111-138, Kenny, A.J. and Turner, A.J., eds., In Mammalian Ectoenzymes, (Elsevier, Amsterdam (1987)).
• 13. Glaser, L. et al., J. Biol. Chem., 242, 1944-1954 (1967).
• 14. Neu, H.C., J. Biol. Chem., 242, 3896-3904 (1967).
• 15. Burns, D.M., and Beacham, I.R., Nuc. Acids. Res., 14, 4325-4342 (1986).
• 16. Gengis-Garber, C., Can. J. Microbiol., 31, 543-548 (1985).
• 17. Tamao, Y., et al., J. Biochem., 109, 24-29 (1991).
• 18. Maznitsa, I.I., et al., FEMS Microbiology Letters 72, 173-176 (1990).
• 19. Misumi, Y., et al., J. Biol. Chem., 265, 2178-2183 (1990).
• 20. Ikehara, Y., et al., Biochimica et Biophysica Acta 470, 202-211 (1977).
• 21. Chen, P.S., et al., Analytical Chem., 28, 1756-1758 (1956).
• 22. US Patent No. 5,110,908.

SEQUENCE LISTING

Claims (12)

Vaccine composition which is an isolated and purified protein of Haemophilus influenzae, wherein said protein the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 or an epitope or epitopes thereof, wherein said vaccine composition has a protective immune response in one Mammalian host triggers. A vaccine composition according to claim 1, wherein said Protein the amino acid sequence, encoded by nucleotides 229 to 2037 of SEQ ID NO: 1. A vaccine composition according to claim 1, wherein the amino acid sequence of amino acids 26-603 of SEQ ID NO: 2 is modified by one or more of the following amino acid residue changes selected from the group consisting of K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H and V436I. A vaccine composition according to claim 1, which also an adjuvant, diluent or carrier includes. A vaccine composition according to claim 4 wherein the Excipient selected is selected from the group consisting of aluminum hydroxide, aluminum phosphate, QS-21, 3-O-deacylated monophosphoryl lipid A and IL-12. Isolated and purified DNA sequence containing a DNA sequence coding for a protein from Haemophilus influenzae or a peptide from said A protein comprising an epitope or epitopes thereof for use as a drug, said protein having an amino acid sequence has, selected out: (i) amino acids 26-603 of SEQ ID NO: 2; (ii) amino acids 26-603 of SEQ ID NO: 2, modified by one or more residual changes, selected from the group K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H and V436I. Isolated and purified DNA sequence according to claim 6 for use as a medicament, said DNA sequence nucleotides 304-2037 of SEQ ID NO: 1, or a DNA sequence, which is assigned thereto under High stringency Southern hybridization hybridizes to standard conditions becomes. Isolated and purified DNA sequence according to either Claim 6 or claim 7 for use in a Haemophilus influenzae A vaccine composition for administration to a mammalian host. Using an isolated and purified DNA sequence, which either for a protein encoded by Haemophilus influenzae, or a peptide of said protein comprising an epitope or epitopes thereof the preparation of a vaccine composition for inducing a protected Immune response Haemophilus influenzae in a mammalian host, said protein an amino acid sequence has, selected out: (i) amino acids 26-603 of SEQ ID NO: 2; (ii) amino acids 26-603 of SEQ ID NO: 2, modified by one or more residual changes, selected from the group K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H and V436I. Isolated and purified protein from Haemophilus influenzae or a peptide of said protein comprising an epitope or epitopes thereof, for use as a medicament, said Protein an amino acid sequence has, selected out: (i) amino acids 26-603 of SEQ ID NO: 2; (ii) amino acids 26-603 of SEQ ID NO: 2, modified by one or more residual changes, selected from the group K79E, N186K, S262G, V294A, E305Q, K327R, T337A, D360Y, R376H and V436I. Isolated and purified protein according to claim 10 or a peptide of said protein comprising an epitope or Epitopes thereof, for use in a Haemophilus influenzae vaccine composition for administration to a mammalian host. Use of an isolated and purified protein according to claim 10 or a peptide of said protein comprising an epitope or epitopes thereof, in the preparation of a vaccine composition to trigger from protective Immune response Haemaphilus influenzae in a mammalian host.